CN104080913B - Modified Helianthi transcription factor can improve yield - Google Patents

Modified Helianthi transcription factor can improve yield Download PDF

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CN104080913B
CN104080913B CN201380007383.9A CN201380007383A CN104080913B CN 104080913 B CN104080913 B CN 104080913B CN 201380007383 A CN201380007383 A CN 201380007383A CN 104080913 B CN104080913 B CN 104080913B
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plant
seqidno
nucleic acid
sequence
polynucleotide
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CN104080913A (en
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R·L·单
D·H·冈萨雷斯
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Bioceres SA
Universidad Nacional del Litoral
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Bioceres SA
Universidad Nacional del Litoral
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention relates to the polynucleotide that encode a kind of modified HaHB4 transcription factor and encode a kind of functional activity fragment of modified HaHB4 transcription factor and the polynucleotide of variant and comprise described polynucleotide and by the carrier of the polypeptide of described polynucleotide encoding and host cell。Present invention additionally comprises the genetically modified host cell of polypeptide and/or the polynucleotide comprising the present invention, plant, seed, pollen and plant part。The present invention farther includes the method producing genetically modified host cell, plant, seed, pollen and plant part and the plant product produced by these transformed hosts。

Description

Modified Helianthi transcription factor can improve yield
The cross reference of related application
This application claims the priority of the U.S. Provisional Application number 61/601,335 that on February 21st, 2012 submits to, it is incorporated herein by reference of text。
About the sequence table submitted to text form
The present invention relates to " sequence table " listed hereinafter, provide with text form。Text file comprises the file (25,010 bytes are created on February 20th, 2013) that title is " HAHB4_SEQID_Listing_ascii_NA.txt ", and it is incorporated herein by reference of text。
Background of invention
Technical field
The present invention relates to the polynucleotide that encode a kind of modified HaHB4 transcription factor and encode a kind of functional activity fragment of modified HaHB4 transcription factor and the polynucleotide of variant and comprise these polynucleotide and by the carrier of the polypeptide of described polynucleotide encoding and host cell。Present invention additionally comprises the genetically modified host cell (including plant cell) of polypeptide and/or the polynucleotide comprising the present invention, plant, seed, pollen and plant part。The present invention farther includes the method producing genetically modified host cell, plant, seed, pollen and plant part and the vegetable products produced by described transformed host。
Background technology
Known homeodomain is present in a class eukaryotic cell transcription factor conservative containing 61 amino acid whose DNA binding motifs, participates in regulating the growth course (Gehring, Science236:1245-1252 (1987)) of higher organism。From the many eukaryotes including fungus, mammal and plant, it has been separated to the coding gene (Gehring etc., Annu.Rev.Biochem.63:487-526 (1994)) containing homeodomain albumen。But, in plant, only it is found that the albumen (being commonly referred to HD-Zip albumen) comprising the homeodomain being connected with interactions between protein leucine zipper motif at present。It has been generally acknowledged that homeodomain-leucine zipper protein participates in regulating growth course (Chan etc., Biochim.Biophys.Acta1442 (1): 1-19 (1998) environment response is relevant to plant；Carabelli etc., PlantJ.4:469-479 (1993)；Schena etc., GenesDev.7:367-379 (1993))。
HaHB4 is a kind of Helianthi (Helianthusannuus) transcription factor, belong to HD-Zip albumen subfamily I and on aminoacid sequence, there is the homogeny of about 50% with the homeodomain of other members in this subfamily, wherein arabidopsis transcription factor AtHB7 and AtHB12 is two exceptions, and it has the homogeny of 60% and 53% respectively with corresponding HaHB4 homeodomain sequence。Under controlled and normal environmental condition, in the sunflower plants of growth, the endogenous expression levels of HaHB4 is very low。It is considered to cause that the toleration that Helianthi plant is coerced for water improves in hydropenia and the rise being exposed under abscisic acid (ABA) condition endogenous HaHB4 and expressing。Similarly, it is reported that the toleration that water is coerced (arid) environment by the transgenic arabidopsis expressing restructuring HaHB4 improves。Specifically, U.S. Patent number 7,674,955 is pointed out, under the same conditions compared with wild-type Arabidopsis plants, the toleration of arid is raised by the transgenic Arabidopsis plants of process LAN HaHB4。But, U.S. Patent number 7,674,955 unexposed modified HaHB4 transcription factor described in the invention, also unexposed under the same conditions compared with wild-type plant, coerce and the output increased of transgenic plant containing these modified HaHB4 transcription factor under non-water stressful environmental at water。
The farm output increased and crop to the tolerance of wider range environmental condition be solve whole world agricultural production encountered provide two kinds of methods by the severe challenge continuing the tilled the land resource shunk and feeding the population continued to increase。Accordingly, it is desirable to provide yield to increase and environment-stress had new crop and the other plant kind of toleration。
Summary of the invention
HaHB4 is the member of a kind of plant transcription factor subfamily, it is believed that participate in regulating growth course environment response is relevant to plant。Have been reported in transgenic plant and can improve the crops toleration for arid and hypersaline environment through the expression of genetically engineered Helianthi transcription factor HaHB4。There is no the report of output increased when standard production, but when expression is too high when HaHB4 is driven by constitutive promoter, have been reported that yield can slightly reduce。Here, we report use modified HaHB4 sequence pair volume increase transgenic plant carry out genetically engineered。More specifically, based on to the order-checking of HaHB4 transgenic in the Semen Tritici aestivi of volume increase, Semen Maydis and Semen sojae atricolor strain, inventor unexpectedly determines, compared to the sequence of natural HaHB4 (SEQIDNO:2), transgenic contains the sequence variation of uniqueness。Additionally, inventor is prepared for another kind of modified HaHB4 sequence, it contains the overlapped different fragments encoding natural and modified HaHB4 albumen。
Correspondingly, in one embodiment, the present invention relates to the polynucleotide encoding a kind of modified HaHB4 transcription factor, compared with the wild-type plant of growth under like environment, coerce or under non-water stress conditions, the expression in transgenic plant of the described polynucleotide all can make yield or the toleration of environment-stress is improved at water。In another embodiment, the present invention relates to the polynucleotide encoding a kind of modified HaHB4 transcription factor, the expression in transgenic plant of the described polynucleotide can accelerate photosynthesis rate。The invention still further relates to the polynucleotide of the functional activity fragment of the polynucleotide of the modified HaHB4 (modHaHB4 (such as mod1HaHB4, mod2HaHB4, mod3HaHB4 and mod4HaHB4)) of coding and coding modHaHB4 and variant and containing these polynucleotide with by the carrier of the albumen of these polynucleotide encodings and host cell。Present invention additionally comprises the genetically modified host cell of the polypeptide containing the present invention and/or polynucleotide (including plant cell), plant, seed, pollen and plant part。Present invention additionally comprises use the polynucleotide of the present invention produce genetically modified host cell, plant, seed, pollen and plant part method and produced by these genetically modified host cells, plant, seed, pollen and plant part seed, offspring and processed vegetable products。
According to an embodiment, the present invention relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 (SEQIDNO:2) fragment or variant, it is characterized in that, compared to the wild-type plant as comparison of growth under like environment, coerce or under non-water stress conditions, the yield of transgenic plant containing described nucleic acid molecules of growth is improved at water。In another embodiment, the present invention relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 (SEQIDNO:2) fragment or variant, it is characterised in that the photosynthesis rate of the transgenic plant containing described nucleic acid molecules is accelerated。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 (SEQIDNO:2) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, coercing or under non-water stress conditions, the yield of transgenic plant containing described nucleic acid molecules of growth is improved at water, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。Another embodiment relates to a kind of nucleic acid molecules, and described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 (SEQIDNO:2) fragment or variant, it is characterised in that the photosynthesis rate of the transgenic plant containing described nucleic acid molecules is accelerated。In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod1HaHB4 (HaHB4.2 (SEQIDNO:4)), it is characterized in that, with under like environment growth as compared with the wild-type plant of comparison, coercing or the output increased of transgenic plant containing described nucleic acid molecules of growth under non-water stress conditions at water。In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod1HaHB4 (HaHB4.2 (SEQIDNO:4)), it is characterized in that, with under like environment growth as comparison wild-type plant compared with, water coerce or under non-water stress conditions growth the transgenic plant comprising described nucleic acid molecules photosynthesis rate accelerate。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod1HaHB4 (HaHB4.2 (SEQIDNO:4)) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, water coerce or under non-water stress conditions growth show output increased containing the transgenic plant of described nucleic acid molecules and/or photosynthesis rate is accelerated, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod1HaHB4 (HaHB4.2 (SEQIDNO:4)) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, coercing or the photosynthesis rate quickening of the transgenic plant containing described nucleic acid molecules of growth under non-water stress conditions at water, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。
In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod2HaHB4 (HaHB4.3 (SEQIDNO:8)), it is characterized in that, with under like environment growth as compared with the wild-type plant of comparison, coercing or the output increased of transgenic plant containing described nucleic acid molecules of growth under non-water stress conditions at water。In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod2HaHB4 (HaHB4.3 (SEQIDNO:8)), it is characterized in that, with under like environment growth as comparison wild-type plant compared with, water coerce or under non-water stress conditions growth containing described nucleic acid molecules transgenic plant photosynthesis rate accelerate。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod2HaHB4 (HaHB4.3 (SEQIDNO:8)) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, coercing or the output increased of the transgenic plant containing described nucleic acid molecules of growth under non-water stress conditions at water, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod2HaHB4 (HaHB4.3 (SEQIDNO:8)) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, coercing or the photosynthesis rate quickening of the transgenic plant containing described nucleic acid molecules of growth under non-water stress conditions at water, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。
In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod3HaHB4 (HaHB4.4 (SEQIDNO:38)), it is characterized in that, with under like environment growth as compared with the wild-type plant of comparison, water coerce or under non-water stress conditions growth show output increased containing the transgenic plant of described nucleic acid molecules and/or photosynthesis rate is accelerated。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod3HaHB4 (HaHB4.4 (SEQIDNO:38)) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, water coerce or under non-water stress conditions growth show output increased containing the transgenic plant of described nucleic acid molecules and/or photosynthesis rate is accelerated, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。
In another embodiment, the nucleic acid molecules of the present invention includes the polynucleotide sequence of a kind of coding HaHB4 variant mod4HaHB4 (HaHB4.5 (SEQIDNO:39)), it is characterized in that, with under like environment growth as compared with the wild-type plant of comparison, water coerce or under non-water stress conditions growth show output increased containing the transgenic plant of described nucleic acid molecules and/or photosynthesis rate is accelerated。Another embodiment relates to a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding mod4HaHB4 (SEQIDNO:39) fragment or variant, it is characterized in that, compared with transgenic HaHB4 (SEQIDNO:2) plant of the same race, water coerce or under non-water stress conditions growth show output increased containing the transgenic plant of described nucleic acid molecules and/or photosynthesis rate is accelerated, the expression of the HaHB4 that wherein recombinates is controlled by same promoter。
Present invention also offers a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence encoding following albumen: (a) HaHB4 variant mod1HaHB4 (HaHB4.2 (SEQIDNO:4))；The functional activity fragment of (b) mod1HaHB4, it is characterised in that the aminoacid sequence of described fragment is not present in corresponding HaHB4 (SEQIDNO:2) sequence；Or the functional activity variant of (c) mod1HaHB4 (SEQIDNO:4), it is characterized in that, described variant comprises the aminoacid sequence with SEQIDNO:4 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and it is characterized in that, the aminoacid sequence of described variant is not present in corresponding HaHB4 (SEQIDNO:2) sequence。Present invention additionally comprises the nucleic acid molecules after separation, described nucleic acid molecules comprises SEQIDNO:3, SEQIDNO:12, SEQIDNO:13, SEQIDNO:15, SEQIDNO:16 or the polynucleotide sequence of its complementary strand and these nucleic acid molecule fragments and variant, and its sequence is not present in corresponding HaHB4 (SEQIDNO:1 and SEQIDNO:18) polynucleotide sequence。
In another embodiment, the invention provides a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence encoding following albumen: (a) HaHB4 variant mod2HaHB4 (HaHB4.3 (SEQIDNO:8))；The functional activity fragment of (b) mod2HaHB4, it is characterised in that the aminoacid sequence of described fragment is not present in corresponding HaHB4 (SEQIDNO:2) sequence；Or the functional activity variant of (c) mod2HaHB4 (HaHB4.3 (SEQIDNO:8)), it is characterized in that, described variant comprises the aminoacid sequence with SEQIDNO:8 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and it is characterized in that, the aminoacid sequence of described variant is not present in corresponding HaHB4 (SEQIDNO:2) sequence。Present invention additionally comprises the nucleic acid molecules after separation, described nucleic acid molecules comprises SEQIDNO:14 or SEQIDNO:17 or the polynucleotide sequence of its complementary strand and these nucleic acid molecule fragments and variant, and its sequence is not present in corresponding HaHB4 (SEQIDNO:1 and SEQIDNO:18) polynucleotide sequence。
In one embodiment, the invention provides a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 variant mod3HaHB4 (HaHB4.4 (SEQIDNO:38))。In another embodiment, the invention provides a kind of nucleic acid molecules, described nucleic acid molecules comprises the polynucleotide sequence of coding HaHB4 variant mod4HaHB4 (HaHB4.5 (SEQIDNO:39))。
According to some embodiments, functional activity polypeptide variants or fragment coded by the polynucleotide of the present invention can in vitro in conjunction with DNA sequence 5'-CAAT (A/T) ATTG-3'(SEQIDNO:11)。In other embodiments, functional activity polypeptide variants coded by the polynucleotide of the present invention or fragment can by 20mMHEPES-NaOH (pH7.6), 50mMKCl, 2mMMgCl at 24 DEG C2, in the solution that forms of 0.5mMEDTA, 1.0mMDTT, 0.5% triton x-100,10% glycerol and 1.0 μ g poly-(dI-dC) in conjunction with DNA sequence 5'-CAAT (A/T) ATTG-3'(SEQIDNO:11)。In other embodiments, express the gene expression profile of the transgenic plant cells of the functional activity polypeptide variants of the present invention or fragment and be different from the wild type control cells (such as parent) being in comparable stage of development and the comparable tissue that is grown in comparable environment。In other embodiments, express the transcriptional profile of the transgenic plant cells of functional activity polypeptide fragment of the present invention or variant and be different from the transgenic HaHB4 cell being in comparable stage of development and the comparable tissue that is grown in comparable environment, and wherein transgenic HaHB4 is controlled by same promoter with modHaHB4 fragment or variant coding sequences。In other embodiments, compared with the wild-type plant of growth under like environment, when water is coerced or grows under non-water stress conditions, express the functional activity polypeptide fragment of the present invention or the output increased of the transgenic plant of variant。In other embodiments, compared with the wild-type plant of growth under like environment, when water is coerced or grows under non-water stress conditions, the photosynthesis rate expressing the functional activity polypeptide fragment of the present invention or the transgenic plant of variant is accelerated。In other embodiments, compared with the transgenic HaHB4 plant of growth under like environment, when water is coerced or grows under non-water stress conditions, expressing the functional activity polypeptide fragment of the present invention or the output increased of the transgenic plant of variant, wherein transgenic HaHB4 is controlled by same promoter with the expression of modHaHB4 fragment or variant coding sequences。In other embodiments, compared with the transgenic HaHB4 plant of growth under like environment, when water is coerced or grows under non-water stress conditions, the photosynthesis rate expressing the functional activity polypeptide fragment of the present invention or the transgenic plant of variant is accelerated, and wherein transgenic HaHB4 is controlled by same promoter with the expression of modHaHB4 fragment or variant coding sequences。
Present invention additionally comprises nucleic acid (including carrier and expression cassette), described nucleic acid comprises the polynucleotide of the present invention being operatively connected with promoter。According to some embodiments, the polynucleotide of the present invention are operatively connectable to constitutive promoter。In certain embodiments, described constitutive promoter is 35SCaMV promoter or Ubi1 promoter。According to other embodiments, the polynucleotide of the present invention are operatively connectable to inducible promoters。In certain embodiments, described inducible promoters is the modified HaHB4 promoter merged with the First Intron of arabidopsis Cox5c-2。
The invention still further relates to the host cell of the nucleic acid comprising the present invention。According to the present invention, the Exemplary host cells used includes but not limited to antibacterial, fungus, insecticide, plant and animal cell。In certain embodiments, described host cell is plant cell。According to some embodiments, described host cell is monocot plant cell。In other embodiments, described host cell is Semen Tritici aestivi (Triticumaestivum), Semen Maydis (Zeamays) or Oryza sativa L. (Oryzasativa)。According to other embodiments, described host cell is dicotyledonous plant cells。In certain embodiments, described host cell is Semen sojae atricolor (Glycinemax)。In other embodiments, described host cell is arabidopsis (Arabidopsisthaliana)。
Present invention additionally comprises the transgenic plant of the polynucleotide sequence comprising the present invention, seed, pollen, plant part and transgenic plant cells。In one embodiment, transgenic plant, seed, pollen, plant part or transgenic plant cells comprise the polynucleotide of coding mod1HaHB4 (SEQIDNO:4)。In other embodiments, the polynucleotide of described coding mod1HaHB4 comprise selected from following sequence of polynucleotide sequence: SEQIDNO:3, SEQIDNO:12, SEQIDNO:13, SEQIDNO:15 and SEQIDNO:16。In another embodiment, transgenic plant, seed, pollen, plant part or transgenic plant cells comprise the polynucleotide of coding mod2HaHB4 (SEQIDNO:8)。In other embodiments, the polynucleotide of described coding mod2HaHB4 comprise the polynucleotide sequence selected from SEQIDNO:14 and SEQIDNO:17。
In another embodiment, transgenic plant, seed, pollen, plant part or transgenic plant cells comprise the polynucleotide sequence of coding mod3HaHB4 (SEQIDNO:38)。In another embodiment, transgenic plant, seed, pollen, plant part or transgenic plant cells comprise the polynucleotide sequence of coding mod4HaHB4 (SEQIDNO:39)。
In another embodiment, the present invention includes a kind of transgenic plant, seed, pollen or plant part, and it comprises the polynucleotide sequence encoding following albumen: mod1HaHB4 (SEQIDNO:4)；The functional activity fragment of (b) mod1HaHB4 (SEQIDNO:4), it is characterised in that the aminoacid sequence of described fragment is not present in the sequence of corresponding HaHB4 (SEQIDNO:2)；Or the functional activity variant of (c) mod1HaHB4 (SEQIDNO:4), it is characterized in that, described variant comprises the aminoacid sequence with SEQIDNO:4 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and it is characterized in that, the aminoacid sequence of described variant is not present in corresponding HaHB4 (SEQIDNO:2) sequence。
In another embodiment, the present invention includes a kind of transgenic plant seed, pollen or plant part, and it comprises the polynucleotide sequence encoding following albumen: (a) mod2HaHB4 (SEQIDNO:8)；The functional activity fragment of (b) mod2HaHB4, it is characterised in that the aminoacid sequence of described fragment is not present in corresponding HaHB4 (SEQIDNO:2) sequence；Or the functional activity variant of (c) mod2HaHB4 (SEQIDNO:8), it is characterized in that, described variant comprises the aminoacid sequence with SEQIDNO:8 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and it is characterized in that, the aminoacid sequence of described variant is not present in corresponding HaHB4 (SEQIDNO:2) sequence。
In another embodiment, the present invention includes a kind of transgenic plant seed, pollen or plant part, and it comprises the polynucleotide sequence encoding following albumen: (a) mod3HaHB4 (SEQIDNO:38)；The functional activity fragment of (b) mod3HaHB4, it is characterised in that the aminoacid sequence of described fragment is not present in corresponding HaHB4 (SEQIDNO:2) sequence；Or the functional activity variant of (c) mod3HaHB4 (SEQIDNO:38), it is characterized in that, described variant comprises the aminoacid sequence with SEQIDNO:38 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and it is characterized in that, the aminoacid sequence of described variant is not present in corresponding HaHB4 (SEQIDNO:2) sequence。
In another embodiment, the present invention includes a kind of transgenic plant seed, pollen or plant part, and it comprises the polynucleotide sequence of coding mod4HaHB4 (SEQIDNO:39)。
According to some embodiments, compared with wild-type plant, express the output increased of the transgenic plant of the polynucleotide of the present invention。In other embodiments, compared with wild-type plant, when water is coerced or grows under non-water stressful environmental, express the output increased of the transgenic plant of the polynucleotide of the present invention。In other embodiments, compared with wild-type plant, when water is coerced or grows under non-water stressful environmental, the photosynthesis rate of the transgenic plant expressing the polynucleotide of the present invention is accelerated。In other embodiment, compared with wild-type plant, in the expression present invention, the transgenic plant of polynucleotide improves for the toleration of one or more environment-stress。In the present invention transgenic plant response and environment-stress that toleration improves includes but not limited to that arid, salinity, osmotic pressure are coerced, low temperature exposes, beat exposure, nitrogen nutrient availability reduce, the minimizing of phosphorus nutrient availability and high plant density。
In other embodiment, compared with the HaHB4 transformed variety of corresponding wild-type plant, expressing the output increased of the transgenic plant of the polynucleotide of the present invention, wherein the expression of the coded polynucleotide of restructuring HaHB4 and the present invention is controlled by same promoter。In other embodiments, compared with the HaHB4 transformed variety of corresponding wild-type plant, when water is coerced or grows under non-water stress conditions, expressing the output increased of the transgenic plant of the polynucleotide of the present invention, wherein the expression of the coded polynucleotide of restructuring HaHB4 and the present invention is controlled by same promoter。In other embodiments, compared with the HaHB4 transformed variety of corresponding wild-type plant, when water is coerced or grows under non-water stress conditions, the photosynthesis rate of the transgenic plant expressing the polynucleotide of the present invention is accelerated, and wherein the expression of the coded polynucleotide of restructuring HaHB4 and the present invention is controlled by same promoter。In other embodiment, compared with the HaHB4 transformed variety of corresponding wild-type plant, the transgenic plant of the polynucleotide of the expression present invention improves for the toleration of one or more environment-stress。
According to some embodiments, described transgenic plant, seed or plant part are monocotyledons。In some embodiments, described transgenic plant, seed or plant part are Semen Tritici aestivi (Triticumaestivum)。In other embodiment, described transgenic plant, seed or plant part are Semen Maydis (Zeamays)。In other embodiments, described transgenic plant, seed or plant part are Oryza sativa L. (Oryzasativa)。In other embodiment, heretofore described transgenic plant, seed or plant part are dicotyledons。In some embodiments, described transgenic plant, seed or plant part are Semen sojae atricolor (Glycinemax)。In other embodiment, described transgenic plant, seed or plant part are arabidopsis (Arabidopsisthaliana)。Present invention additionally comprises the seed of transgenic plant cells and transgenic plant and offspring, and the seed that produces of these transgenic plants, its seed and its offspring and processed vegetable products。
The invention still further relates to the method producing transformed host, described transformed host includes but not limited to the antibacterial of polynucleotide, fungus and the plant cell and the transgenic plant that comprise the present invention。The genetically modified host cell produced according to these methods and the offspring of transgenic plant and seed, and the processed vegetable products that these transformed hosts, its seed and its offspring produce。
Brief Description Of Drawings
Figure 1A provides the sequence alignment between HaHB4 transcription factor (SEQIDNO:2), modified HaHB4 transcription factor 1 (mod1HaHB4/HaHB4.2 (SEQIDNO:4)) and modified HaHB4 transcription factor 2 (mod2HaHB4 (SEQIDNO:8))。Square frame shows homeodomain-Leucin-zipper domains。Figure 1B provides the sequence alignment between HaHB4 transcription factor (SEQIDNO:2), mod1HaHB4 (HaHB4.2 (SEQIDNO:4)), mod2HaHB4 (HaHB4.3 (SEQIDNO:8)), mod3HaHB4 (HaHB4.4 (SEQIDNO:38)) and mod4HaHB4 (HaHB4.5 (SEQIDNO:39))。
Fig. 2 A-2J provides the exemplary carrier of the polynucleotide transformed host cell for using the present invention and the schematic diagram of structure。Fig. 2 A-C respectively describes structure pGmHaHB4.2, pGmPrHB4.2 and pGmPrInHB4.2 for soybean transformation and other plant。Fig. 2 D-G respectively describes for the structure pTaBAR of transformed wheat and other plant, pTaHaHB4.2, pTaPrHB4.2 and pTaPrInHB4.2。Fig. 2 H-J respectively describes structure pZmHaHB4.2, pZMPrHB4 and pZmPrInHB4.2 for maize transformation and other plant。Abbreviation: aadA (the aminoglycoside 3'-adenosyl transferase gene of Shigella flexneri (S.flexneris) 2a, produce the resistance to streptomycin), bar (the phosphine oxamate acetyl transferase gene from streptomyces hygroscopicus (S.Hygroscopicus), produce the resistance to herbicide glufosinate-ammonium and derivant thereof), P35S (cauliflower mosaic virus 35 S promoter), Tnos (NOS-Term；The 3' terminator of Agrobacterium (A.tumefaciens) rouge alkali synthetase gene)；Tvsp (soybean nutritional storage protein gene 3' terminator), TEV (transcriptional enhancer of tobacco etch virus)；RB (the T-DNA right margin fragment of Agrobacterium (A.tumefaciens) nopaline bacterial strain)；LB (the T-DNA left margin fragment of Agrobacterium (A.tumefaciens) nopaline bacterial strain)；PVS1 (the wide host range plasmid of pseudomonas (Pseudomonas))；AmpR (ampicillin resistance gene)；KanR (kalamycin resistance gene)；Ori (origin of replication in antibacterial)；HaHB4.2 (HaHB4 sequence (pTHaHB4.2c insertion) modified described in embodiment part)；LPF (the HaHB4 promoter with modifying described in embodiment)；And COX5c-2 intron (First Intron of COX5c-2)。
Block diagram in Fig. 3 A-3D shows the raising of yield in the genetically modified crops that (are not intended to supply water, be under non-water stress conditions) in high yield environment when the field through irrigating。Transgenic corns data described by Fig. 3 A-3B show two kinds of homozygous transgenic Semen Maydis strains (constitutive expression HaHB4.2 (SEQIDNO:4)) and the corresponding grain yield (kg/ha) of wild type control Semen Maydis (WT)。Data acquisition is from the parallel test point in two soil type areas: receives the sandy loam (Fig. 3 A) of 626mm rainfall between planting season and receives the well-drained sandy loam (Fig. 3 B) of 545mm rainfall in the crop cycle。Transgenic wheat data described by Fig. 3 C show a kind of homozygous transgenic lines (constitutive expression HaHB4.2 (SEQIDNO:4)) and the corresponding grain yield (kg/ha) of wild type control Semen Tritici aestivi (WT)。Data acquisition in Fig. 3 C is from the parallel test point of well-drained sandy loam area (pH7.14%, OM1.57%)。Use supplement irrigation to provide the water of 755mm within the crop cycle。Genetically engineered soybean data described by Fig. 3 D show two kinds of homozygous transgenic Semen sojae atricolor strains (constitutive expression HaHB4.2 (SEQIDNO:4)) and the corresponding grain yield (kg/ha) of wild type control Semen sojae atricolor (WT)。Data acquisition in Fig. 3 D is from the parallel test point of well-drained sandy loam area (pH5.99%, OM1.41%)。Use supplement irrigation to provide the water of 579mm within the crop cycle。
Block diagram in Fig. 4 shows the trans-activating factor activity of multiple modified HaHB4 in a simple yeast crossbreeding test。
The block diagram of Fig. 5 show two uses converted containing and 2 independent experiments carrying out of the arabidopsis thaliana of the expression cassette (such as, 35S.H4.2) of 35S constitutive promoter that is operatively connected of the nucleotide sequence of coding mod1HaHB4 (HaHB4.2 (SEQIDNO:4)) in the photosynthesis rate that records。
Detailed Description Of The Invention
Definition
Except as otherwise noted, the usual implication that all scientific and technical terminologies used herein are understood with one skilled in the art of the present invention is identical。In case of conflict, it is as the criterion with the application (including definition)。Additionally, unless the context otherwise requires, singular references should include plural number and plural term should include odd number。All publications, patent and other lists of references addressed herein are all incorporated herein by reference of text。
Term " polynucleotide " includes any one or more nucleic acid fragments (such as DNA or RNA fragment) existed in single core acid molecule and nucleic acid molecules, and represent separated nucleic acid molecules or build (such as, carrier) (such as the messenger RNA (mRNA) relevant to the polynucleotide of the present invention or plasmid DNA (pDNA))。Term " nucleic acid " and " polynucleotide " are used interchangeably herein。" separated " nucleic acid or polynucleotide refer to the nucleic acid molecules, DNA or RNA that extract from its natural surroundings。Such as, purification (partially or substantially purification) polynucleotide in separated polynucleotide include being present in Heterologous Host Cells recombination of polynucleotide or solution。Separated RNA molecule includes the inner or in vitro RNA transcript of the polynucleotide of the present invention。Additionally, polynucleotide or nucleic acid can be maybe to include controlling element, such as promoter, ribosome binding site or transcription terminator。
Term used herein " coding region " refers to by being translated into the molecular nucleic acid molecules of amino acid whose password。Although " termination codon " (TAG, TGA or TAA) does not translate into aminoacid, but it is believed that it is a part for coding region, and any flanking sequence such as promoter, ribosome binding site, transcription terminator, intron etc. are not all parts for coding region。Additionally, the carrier of the present invention, polynucleotide or nucleic acid codified heterologous coding region。
In some embodiments, described polynucleotide or nucleic acid are DNA。When DNA, the polynucleotide comprising polypeptide encoding nucleic acid comprise promoter and/or other transcribes or translate control element, and these elements and one or more coding regions are operatively connected。Being operatively connected finger when the coding region of gene outcome (such as polypeptide) is connected in this way with one or more regulating and controlling sequences, the expression of this gene outcome is placed under impact or the control of this regulating and controlling sequence。If the mRNA of gene outcome transcribes needed for the induction of promoter function makes coding, if and the attribute connected between two DNA fragmentations do not disturb expression regulation sequence instruct gene product expression or do not disturb the ability of transcription DNA template, then two DNA fragmentations (such as polypeptid coding area and connected promoter thereof) " being operatively connected " or " operable be connected "。Therefore, if promoter can affect transcribing of nucleic acid, then promoter region and polypeptide encoding nucleic acid are operatively connected。Generally, the polynucleotide sequence that operable connected expression is connected is adjacent and can connect two adjacent protein encoding regions if desired in reading frame to form " fusion protein "。" fusion protein " is the albumen of a kind of aminoacid sequence comprising and being derived from two or more heterologous polypeptides。
Term used herein " promoter " refers to that one section of function is the regulatory nucleic acid fragment controlling one or more genetic transcriptions, it is positioned at the upstream (relative to transcriptional orientation) of the transcriptional start site of gene, and structurally comprise binding site (transcriptional start site) and other DNA sequence of DNA RNA polymerase relied on, include but not limited to Binding site for transcription factor, suppress son and activator protein binding site and known in the art direct or indirect from any other polynucleotide sequence of the promoter regulation amount of transcribing。
" plant promoter " be a kind of can in plant cell the promoter of initiation transcription, regardless of whether whether its source is plant cell, for instance known Agrobacterium promoter can play a role in plant。Correspondingly, plant promoter includes the promoter DNA sequence that derives from plant, plant virus and antibacterial (such as Agrobacterium)。Grow the promoter example controlled only to include or preferably in the promoter of initiation transcription in some cell type or tissue (such as leaf, root or seed)；And the promoter of " can induce " or " can suppress " (including such as anaerobic environment, being exposed to some chemicals and be exposed to the change of light application time) under environmental condition。The promoter of the present invention includes " non-composing type " or " can induce " promoter of " composing type " promoter active in majority tissue and physiology (such as by some compound of exogenous use) or developmental regulation under most physiology and developmental condition。The example of constitutive promoter disclosed herein includes but not limited to 35SCaMV promoter (referring to such as pGmHaHB4.2 (Fig. 2 A) and pZmHaHB4.2 (Fig. 2 H)) and Ubi promoter (referring to such as pTaHaHB4.2 (Fig. 2 E))。There is disclosed herein the example of the inducible promoters used in the present invention, include but not limited to that modified HaHB4 promoter (referring to such as pGmPrHB4.2 (Fig. 2 B), pTaPrHB4.2 (Fig. 2 F) and pZMPrHB4.2 (Fig. 2 I)) and the First Intron by modified HaHB4 promoter with arabidopsis Cox5c-2 gene merge the inducible promoters formed (referring to such as pGmPrInHB4.2 (Fig. 2 C), with pTaPrInHB4.2 (Fig. 2 G), and pZmPrInHB4.2 (Fig. 2 J))。
" regulating and controlling sequence " or " controlling element " refer to be positioned at coded sequence upstream (5 ' non-coding sequence), within or the nucleotide sequence of downstream (3 ' non-coding sequence), it affects the transcribing of related coding sequences, RNA processing or stability or translation。Regulating and controlling sequence can comprise promoter, enhancer, manipulation, suppress son, transcription stop signals, translation targeting sequencing, intron, polyadenylic acid recognition sequence, RNA Processing position, effector binding site and loop-stem structure。
Term " plasmid ", " carrier " and " expression cassette " can exchange use in this article and represent a kind of generally with the extra chromatin element not being cell centre metabolism part。These elements can be autonomous replication sequence, gene integration sequence, phage or nucleotide sequence, the strand of linear or ring-type or double-stranded DNA or RNA, being derived from any source, wherein a large amount of nucleotide sequences have connected or have recombinated into can by the unique texture of the promoter fragment of selected genes product and DNA sequence with suitable 3 ' untranslated sequences introducing cell。For the compositions in the method in the application present invention and the preparation present invention, the carrier (including plasmid, cosmid, phage or Agrobacterium binary vector) of any double-strand or strand, linear or annular form can be used, it can be may not be autonomous transfer or moveable, it is possible to converts protokaryon or eucaryon host by being integrated into the mode of cellular genome or is present in chromosome outer (the automatic plasmid replication as with replication origin)。Specifically including shuttle plasmid, what represent a kind of natural or design can at the DNA vector of two kinds of different hosts (being selected from such as antibacterial, higher plant, yeast or fungal cell) internal duplication。
Or, term " expression cassette " can represent the nucleic acid fragment expressing one or more specific proteins, including the regulating and controlling sequence of (5' non-coding sequence) before albumen coded sequence and (3' terminator sequence) afterwards。
Term used herein " conversion " refers to transfer in host organisms by nucleic acid, causes the heredity with hereditary stability。Host organisms containing transformed nucleic acid fragment is called " transgenic " or " restructuring " or " conversion " organism。
Term used herein " expression " represents the justice (mRNA) of nucleic acid fragment or the transcribing and accumulating of antisense RNA that are derived from the present invention。Expression can also refer to that mRNA translation forms polypeptide。This process includes any embodiment of the functional existence of the polynucleotide of cell inner expression, gene or polypeptide, includes but not limited to gene knockout and transient expression and stably express。
Term used herein " polypeptide " is intended to include singulative " polypeptide " and plural form " polypeptide ", refers to the molecule that the monomer (aminoacid) by amido link (also referred to as peptide bond) linearly connected forms。Term " polypeptide " refers to that any one or more by two or more amino acids formed chains, and does not indicate that the concrete length of the post translational modification type of product or product。Term " polypeptide " and " albumen " are used interchangeably herein。
Term used herein " host cell " represents can through any kind of cell system of the genetic engineering modified polypeptide to express the present invention。Host cell includes cultured cells, yeast cells, insect cell, plant cell and mammalian cell etc. and the cell comprised in transgenic organism (plant tissue such as transgenic plant or cultivation)。
Term used herein " plant " includes whole plant, plant part (such as leaf, stem, root etc.), seed, pollen and plant cell and offspring thereof。Plant cell herein includes but not limited to seed, suspension culture, embryo, meristematic regions, corpus callosum, leaf, root, branch, gamete, spore, pollen and sporidiole。
" transgenic plant cells " used herein represents the polynucleotide sequence of the inverted present invention with stable integration or with comprising the plant cell of the automatic replicating vector of polynucleotide sequence in the present invention。Term used herein " stable integration " represents that being integrated in chromatin, heredity stablizes and can pass through the polynucleotide that subsequent generation is hereditary。The method of conversion plant cell described herein or known in the art and its include but not limited to the coated microparticle bombardment of Agrobacterium-medialed transformation, electroporation and DNA。Transgenic plant cells in the present invention includes the middle progeny plants cell existed of the plant cell of initial conversion, regeneration and/or differentiated tissue's (as with transgenic plant stable integration, non-natural recombination of polynucleotide sequence in the present invention) existed with cell culture or organism form。Transgenic plant cells also includes the seed or the pollen that derive from transgenic plant offspring。For purposes of the present invention, term " corn ", " seed " and " grain " is used interchangeably。
" transgenic plant " used herein includes the plant that genome is contained within the polynucleotide of the present invention。Generally, the polynucleotide stable integration of the present invention enters the genome of host, so that described polynucleotide can be transferred to subsequent generation。Described heterologous polynucleotide can be partially integrated into genome individually or as recombinant expression cassettes。" transgenic " used herein includes the cell of any polynucleotide containing the present invention, cell line, corpus callosum, plant part or plant, including being initially the Transgenics so changed and passing through Transgenics produced by sexual hybridization or asexual propagation from initial Transgenics。
Term used herein " functional activity " represents polynucleotide and the polypeptide (including fragment and variant) of at least one functional activity that can present HaHB4 or modified HaHB4 transcription factor。In one embodiment, the functional activity polypeptide in the present invention (including fragment and variant) can in vitro in conjunction with DNA sequence 5'-CAAT (A/T) ATTG-3'(SEQIDNO:11)。In other embodiment, the functional activity polypeptide of the present invention can by 20mMHEPES-NaOH (pH7.6), 50mMKCl, 2mMMgCl at 24 DEG C2, in the solution that forms of 0.5mMEDTA, 1.0mMDTT, 0.5% triton x-100,10% glycerol and 1.0 μ g poly-(dI-dC) in conjunction with DNA sequence 5'-CAAT (A/T) ATTG-3'(SEQIDNO:11)。For identifying that the DNA binding tests by the functional activity polypeptide of the polynucleotide encoding of the present invention is as known in the art, and conventional can apply or improve on demand。According to an embodiment, what can use synthetic comprises sequence 5'-CAAT (A/T) ATTG-3'(SEQIDNO:11) double chain oligonucleotide carry out electrophoretic mobility shift assay (EMSA), for measurement function modify HaHB4DNA associated proteins whether exist。Such as, can by antibacterial (such as the escherichia coli) albumen after purification or the plant cell nucleoprotein after purification with containing sequence 5'-AATTCAGATCTCAATAATTGAGAG-3'(SEQIDNO:36) and radioactive label double-stranded DNA 5'-GATCCTCTCAATTATTGAGATCTG-3'(SEQIDNO:37) at binding medium (containing 20mMHEPES-NaOH (pH7.6), 50mMKCl, 0-2mMEDTA, 1.0mM dithiothreitol, DTT (DTT), 0.5% triton x-100, 20% glycerol and 1.0ug poly-(dI-dC)) in hatch at 25 DEG C 20 minutes, supplement addition 2.5% (w/v) Ficoll, subsequently reactant it is loaded in acrylamide gel and evaluates the change of DNA mobility in gel, with this crop DNA result combined。Other evaluation transcription factor are combined with DNA sequence motif and technology and the test of protein and DNA binding affinity are known in the art。
In another embodiment, the functional activity polypeptide of the present invention is different from the endogenous host plant protein of HaHB4 transcription factor (SEQIDNO:2) in conjunction with one or more in physiological conditions at (including fragment and variant)。In some embodiments, the functional activity polypeptide of the present invention can in conjunction with the endogenous host plant protein not being combined with the corresponding polypeptide part of HaHB4。In other embodiments, the functional activity polypeptide of the present invention can not in conjunction with the endogenous host plant protein being combined with the corresponding polypeptide part of HaHB4。Determine that the method for protein-protein interaction and material (including such as yeast two-hybrid system) are to it known in the art, and conventional can be suitable for and apply the protein-protein interaction between the polypeptide to evaluate the present invention。In one embodiment, the functional activity polypeptide of the present invention is different from the arabis protein of HaHB4 transcription factor (SEQIDNO:2) in conjunction with one or more in physiological conditions at (including fragment and variant)。Protoplasts of Arabidopsis thaliana broken by ultrasonic double cross (P2H) system (being disclosed in: Ehlert etc., PlantJ.46:890-900 (2006)) is included for analyzing the illustrative methods of protein-protein interaction between the polypeptide of the present invention and arabis protein。
In other embodiment, functional activity polynucleotide or the host cell of polypeptide (including fragment and variant) containing the present invention present the transcriptional profile different from the compared with control cells of the polynucleotide without the present invention or polypeptide (such as wild-type genotype)。In the embodiment of, under arid, high salt conditions or ethylene expose, functional activity polynucleotide or the host cell of polypeptide containing the present invention present the transcriptional profile different from the compared with control cells (such as wild-type genotype) without polynucleotide in the present invention or polypeptide。
Reagent known in the art and technology can be used to carry out transcriptional profile analysis。These technology include but not limited to that the protection of microarray, RT-PCR, RNase, Northern and Western analyze。Generally expressed, by what microarray analysis carried out, the change that profile analysis can be used for measuring multiple heterogeneic differential expression or induction generation simultaneously。Microarray analysis technology and other technology (protect such as Northern, Western, PCR and RNase and analyze) being used for evaluating coded sequence expression level are (Schena etc., Science270:467-470 (1995) known in the art；Baldwin etc., Curr.Opin.PlantBiol.2 (2): 96-103 (1999)；DangondF, Physiol.Genomics2:53-58 (2000)；VanHal etc., J.Biotechnol.78:271-280 (2000)；Richmond and Somerville, Curr.Opin.PlantBiol.3:108-116 (2000)；Almoguera etc., PlantMol.Biol.19:781-792 (1992)；And the chief editor such as Ausubel, 1998, " molecular biology test guide " (CurrentProtocolsinMolecularBiology), New York John Wei Lisen publishing house (john wiley & sons))。
In one embodiment, described transcriptional profile analysis carries out in the transgenic Arabidopsis plants of the present invention, plant part or plant cell。Illustrative methods for the transcriptional profile analysis of the transgenic plant of the present invention, plant part or plant cell sees Manavella etc., PlantJournal48:125-137 (2006) and Hilson etc., Gen.Res.14:2176-2189, above-mentioned document is incorporated herein each through incorporated。Such as, according to a kind of method, the CATMA array containing 24,576 gene specific labels from arabidopsis is used to carry out transcriptome analysis。In other method, the oligonucleotide primers designed by the sequence according to public channel acquisition is used to carry out real-time PCR (referring to such as Arabidopsis.org；With Crowe etc., Nucl.Acids.Res.31:156-158 (2003))。
In another embodiment, compared with the matched group (such as wild type) without polynucleotide in the present invention or polypeptide, what functional activity polynucleotide containing the present invention or the host cell of polypeptide showed different mRNA or protein transcribes or expresses the group that overview, described mRNA or protein select free LOX2, CSD1, ERF2, ERF5, ACO, SAM, EIN1 and EIN3 to form。In another embodiment, expressing under controlling with same promoter compared with the transgenic HaHB4 host cell of HaHB4 (SEQIDNO:2), functional activity polynucleotide or the host cell of polypeptide (including fragment and variant) containing the present invention show different transcriptional profile。In another embodiment, express compared with the transgenic HaHB4 host cell of HaHB4 (SEQIDNO:2) under controlling with same promoter, under drought condition, high salt conditions or ethylene exposure condition, functional activity polynucleotide or the host cell of polypeptide containing the present invention show different transcriptional profile。In another embodiment, express compared with the transgenic HaHB4 host cell of HaHB4 (SEQIDNO:2) under controlling with same promoter, functional activity polynucleotide or the host cell of polypeptide containing the present invention present different mRNA or protein expression profile, described mRNA or protein and select the group of free LOX2, CSD1, ERF2, ERF5, ACO, SAM, EIN1 and EIN3 composition。
In another embodiment, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide or the transgenic plant of polypeptide (including fragment and variant) containing the present invention show different yield。In another embodiment, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide or the transgenic plant of polypeptide (including fragment and variant) containing the present invention show different photosynthesis rates。In a specific embodiment, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide containing the present invention or the output increased of the transgenic plant of polypeptide (including fragment and variant)。In a specific embodiment, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide or the photosynthesis rate of the transgenic plant of polypeptide (including fragment and variant) containing the present invention are accelerated。In a specific embodiment, under identical non-water stress conditions, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide containing the present invention or the output increased of the transgenic plant of polypeptide (including fragment and variant)。In a specific embodiment, under identical non-water stress conditions, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide or the photosynthesis rate of the transgenic plant of polypeptide (including fragment and variant) containing the present invention are accelerated。
In another embodiment, expressing under controlling with same promoter compared with the transgenic plant of HaHB4 (SEQIDNO:2), functional activity polynucleotide or the transgenic plant of polypeptide (including fragment and variant) containing the present invention present different yield。In another embodiment, expressing under controlling with same promoter compared with the transgenic plant of HaHB4 (SEQIDNO:2), functional activity polynucleotide or the transgenic plant of polypeptide (including fragment and variant) containing the present invention present different photosynthesis rates。
In a specific embodiment, express compared with the transgenic plant of HaHB4 (SEQIDNO:2) under controlling with same promoter, functional activity polynucleotide containing the present invention or the output increased of the transgenic plant of polypeptide。In a specific embodiment, expressing compared with the transgenic plant of HaHB4 (SEQIDNO:2) under controlling with same promoter, functional activity polynucleotide or the photosynthesis rate of the transgenic plant of polypeptide containing the present invention are accelerated。
In some embodiments, under identical water condition and same promoter control, output increased compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), when functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide grow under non-water stress conditions。In some embodiments, under identical water condition and same promoter control, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), photosynthesis rate when functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide grow under non-water stress conditions is accelerated。In some embodiments, under same water condition and same promoter control, output increased compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), when functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide grow under water stress conditions。In some embodiments, under identical water condition and same promoter control, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), photosynthesis rate when functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide grow under water stress conditions is accelerated。
In other embodiment, same promoter control with under identical water condition, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide water coerce with non-water stress conditions under grow time output increased。In other embodiment, same promoter control with under identical water condition, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide water coerce with non-water stress conditions under grow time photosynthesis rate accelerate。
In other embodiments, under identical water condition, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide containing the present invention or the transgenic plant of polypeptide (including fragment and the variant) output increased when water is coerced or grows under non-water stress conditions。In other embodiments, under identical water condition, compared with the matched group of the polynucleotide without the present invention or polypeptide (such as wild type genotype), functional activity polynucleotide or the transgenic plant of polypeptide (including fragment and the variant) photosynthesis rate when water is coerced or grows under non-water stress conditions containing the present invention are accelerated。In a specific embodiment, same promoter control with under identical water condition, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), functional activity polynucleotide containing the present invention or the transgenic plant of the polypeptide output increased when water is coerced or grows under non-water stress conditions。In a specific embodiment, same promoter control with under identical water condition, compared with the transgenic plant expressing HaHB4 (SEQIDNO:2), functional activity polynucleotide or the transgenic plant of the polypeptide photosynthesis rate when water is coerced or grows under non-water stress conditions containing the present invention are accelerated。
Detailed description of the invention is directed to use with the polynucleotide of the present invention method to produce plant, described plant is in does not restrict water supply under condition, under condition of restricting water supply or do not restrict water supply condition and output increased when restricting water supply under condition, described method comprises the steps of and the polynucleotide of the present invention is imported host plant cell, polynucleotide molecule is selected to exist to generate transgenic plant cells, and from transgenic plant cells regeneration transgenic plant, so that the yield that transgenic plant is under do not restrict water supply condition or condition of restricting water supply is higher than comparable wild-type plant or transgenic HaHB4 plant, wherein HaHB4 is under the control of same promoter。Detailed description of the invention is directed to use with the polynucleotide of the present invention method to produce plant, described plant is in does not restrict water supply under condition, under condition of restricting water supply or do not restrict water supply condition and when restricting water supply under condition photosynthesis rate accelerate, described method comprises the steps of and the polynucleotide of the present invention is imported host plant cell, polynucleotide molecule is selected to exist to generate transgenic plant cells, and from transgenic plant cells regeneration transgenic plant, so that the photosynthesis rate that transgenic plant is under do not restrict water supply condition or condition of restricting water supply is higher than comparable wild-type plant or transgenic HaHB4 plant, wherein HaHB4 is under the control of same promoter。
For example, for nucleotide sequence and the present invention with reference to nucleotide sequence at least 95% " identical " nucleic acid molecules or polynucleotide for, may comprise up to except 100 nucleotide every in polynucleotide sequence five relative to the point mutation of canonical sequence except, the nucleotide sequence of polynucleotide molecule is identical with canonical sequence。In other words, the polynucleotide identical with reference to nucleotide sequence at least 95% in order to obtain nucleotide sequence, can delete or replace in canonical sequence the nucleotide up to 5% with other nucleotide, or the nucleotide that length can be up to canonical sequence total nucleotide 5% is inserted in canonical sequence。
It practice, typically via known computer program determine the nucleotide sequence of any specific nucleic acid molecule or polypeptide and the present invention or peptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical。Term known in the art " same percentage " refers to, by the relation between the determined two or more peptide sequences of sequence alignment or two or more polynucleotide sequence。In this area, " homogeny " also refers to the sequence degree of correlation between polypeptide or polynucleotide sequence, is optionally determined by the coupling between this type of sequence string。Known method can be used to readily calculate " homogeny " and " similarity ", and described method includes but not limited to the following stated: 1.)Computational MolecularBiology (" computational molecular biology ")(Lesk, A.M. edit) Oxford University Press (OxfordUniversity): New York (1988)；2.)Biocomputing:InformaticsandGenomeProjects is (" raw Thing calculates: informatics and genome plan ")(Smith, D.W. edit) academic press (Academic): New York (1993)；3.)ComputerAnalysisofSequenceData, the PartI (" computer of sequence data Analyze ", Part I)(Griffin, A.M. and Griffin, H.G. edit) mankind publishing house (Humania): New Jersey (1994)；4.)The SequenceAnalysisinMolecularBiology (" sequence in molecular biology Row are analyzed ")(vonHeinje, G. edit) academic press (Academic) (1987)；And 5.)Sequence AnalysisPrimer (" sequence analysis primer ")(Gribskov, M. and Devereux, J. edit) Stockton Press (Stockton): New York (1991)。
Devise for the method for optimizing measuring homogeny to complete best coupling between cycle tests。The method measuring homogeny and similarity encodes in publicly available computer program。The calculating of sequence alignment and percent identities can use LASERGENE bioinformatics to calculate the MegAlign of external memberTMProgram (the DNASTAR company (DNASTARInc.) of state of Wisconsin Madison) is implemented。The multiple ratio of sequence is to using " the Clustal method of sequence alignment ", it comprises many algorithms, including " the ClustalV method of sequence alignment ", corresponding to the comparison method of ClustalV labelling (referring to Higgins and Sharp, CABIOS5:151-153 (1989)；Higgins etc., Comput.Appl.Biosci., 8:189-191 (1992)), and the MegAlign of external member can be calculated in LASERGENE bioinformaticsTMProgram finds (DNASTAR company (DNASTARInc.))。For multiple ratio pair, default value is relative to Gap Penalty (GAPPENALTY)=10 and Gap Length Penalty (GAPLENGTHPENALTY)=10。Using the default parameters that Clustal method carries out paired comparison and the calculating of protein sequence same percentage is K tuple (KTUPLE)=1, Gap Penalty (GAPPENALTY)=3, window (WINDOW)=5 and diagonal retain (DIAGONALSSAVED)=5。For nucleic acid, these parameters are K tuple (KTUPLE)=2, Gap Penalty (GAPPENALTY)=5, and window (WINDOW)=4 and diagonal retain (DIAGONALSSAVED)=4。After using ClustalV program to carry out sequence alignment, by checking that " sequence distance " table in same program can obtain " same percentage "。In addition it is possible to use " the ClustalW method of sequence alignment ", it corresponds to the comparison method being labeled as ClustalW (referring to Higgins and Sharp, CABIOS.5:151-153 (1989)；Higgins etc., Comput.Appl.Biosci.8:189-191 (1992)), and the MegAlign of external member can be calculated in LASERGENE bioinformaticsTMV6.1 program finds (DNASTAR company (DNASTARInc.))。Default parameters (the Gap Penalty (GAPPENALTY)=10 of multiple ratio pair, Gap Length Penalty (GAPLENGTHPENALTY)=0.2, postpone the scattered sequence (DelayDivergenSeqs) (%)=30 that becomes, DNA changes proportion (DNATransitionWeight)=0.5, protein proportion matrix (ProteinWeightMatrix)=Gonnet series (GonnetSeries), DNA proportion matrix (DNAWeightMatrix)=IUB)。After using ClustalW program to carry out sequence alignment, by checking that " sequence distance " table in same program can obtain " same percentage "。
For example, for the polypeptide of inquiry aminoacid sequence at least 95% " identical " in aminoacid sequence and the present invention, may comprise up to five except 100 aminoacid every in subject polypeptide sequence relative to except the aminoacid change inquiring about aminoacid sequence, the aminoacid sequence of subject polypeptide is identical with inquiry polypeptide。In other words, having at least 95% identical polypeptide for obtaining aminoacid sequence with inquiry aminoacid sequence, the amino acid residue being up to 5% in object sequence can be inserted into, lack or use other aminoacid replacement。Can there is any position between amino or c-terminus or these end positions with reference to aminoacid sequence in these changes of canonical sequence, it is possible to be dispersed on canonical sequence, or in the one or more adjacent group in canonical sequence。
It practice, generally use known computer program determine any specific polypeptide and have at least 80% with reference to peptide sequence, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical。Determine that between search sequence (certain sequence of the present invention) and object sequence, a method of best overall match is to adopt based on Brutlag etc., the FASTDB computer program of the algorithm of Comp.App.Biosci.6:237245 (1990) is determined, the method is also referred to as overall sequence alignment。In sequence alignment, inquiry and object sequence or be all nucleotide sequence, or be all aminoacid sequence。The result of described overall sequence alignment is expressed as same percentage。In FASTDB amino acid alignment, preferred parameter is: matrix (Matrix)=PAM0, k-tuple (k-tuple)=2, Mismatch Penalty (MismatchPenalty)=1, connect point penalty (JoiningPenalty)=20, random packet length (RandomizationGroupLength)=0, block score value (CutoffScore)=1, window size (WindowSize)=sequence length, Gap Penalty (GapPenalty)=5, notch size point penalty (GapSizePenalty)=0.05, shorter one in the length of window size (WindowSize)=500 or object aminoacid sequence。
If object sequence is shorter than search sequence is owing to N-or C-end lacks but not the disappearance of centre, it is necessary to result is carried out manual synchronizing。This is because FASTDB program when calculating overall same percentage it is not intended that N-and the C-end of object sequence blocks。Relative search sequence is had to the object sequence blocked at N-and C-end, correct same percentage by calculating in search sequence at object sequence N-and C-end the percent not accounting for search sequence total bases with the residue number of corresponding object residue match/align。Determine whether residue mates/align by the result of FASTDB sequence alignment。Then adopted special parameter to calculate in gained same percentage by above-mentioned FASTDB program and deduct this percentage value, it is thus achieved that final same percentage score。Namely this final same percentage score is used to the purpose of the present invention。In order to manually adjust same percentage score, only consider the residue that object sequence N-and C-end do not mate with search sequence/align。It is to say, the resi-dues of only query object sequence N-and C-terminal residue distal exterior。
Such as, by have the object sequence of 90 amino acid residues and have 100 residues search sequence comparison to determine same percentage。There is the N-end in object sequence in disappearance, therefore FASTDB alignment does not show the coupling/alignment of initial 10 residues of N-end。These 10 are not matched residue and represent 10% total residue number of residue number/search sequence (N-and the C-end do not mate) of sequence, therefore need to deduct 10% from the same percentage that FASTDB program computation obtains。If all the other 90 residues are perfect match, then final same percentage will be 90%。In another example, the object sequence of 90 residues and the search sequence of 100 residues are compared。Current disappearance is inside disappearance, therefore N-or the C-end of object sequence not have not with inquire about the residue matching/aliging。In this situation, FASTDB calculates gained same percentage without manual synchronizing。As it has been described above, only to being shown as in FASTDB comparison outside object sequence N-and C-end, the resi-dues not mating with search sequence/aliging carries out manual synchronizing。For the object of the invention, it is not necessary to carry out other manual synchronizing。
It is used as blast program (default parameters in use program) available in NCBI (NationalCenterforBiotechnologyInformation, NCBI) and determines the same percentage of polynucleotide and/or polypeptide。
After term used herein " same percentage " represents optimization comparison, do not have discrepant degree by between two DNA or protein fragments that component (such as nucleotide sequence or aminoacid sequence) comparison window is observed。Cycle tests is total divided by the sequence component in reference fragment with canonical sequence common same composition number of sequence of two arrangement post-fragment on the arrangement window that " the identical mark " of comparison post-fragment is less equal in total length cycle tests or total length canonical sequence。" same percentage " (" identical % ") is multiplied by 100 equal to identical mark。This kind of optimization comparison should be interpreted as the Local Alignment of DNA sequence。For protein comparison, the Local Alignment of protein sequence should allow to add breach to realize optimization comparison。After the comparison that the calculating of same percentage is based on, sequence length does not comprise the breach introduced in every sequence by comparison。
Term used herein " variant " polynucleotide or polypeptide represent and use such as recombinant DNA technology that amino acid residue and polynucleotide site are inserted respectively, lack, suddenly change and replaced, thus the polynucleotide different from the polynucleotide specifically noted in the present invention or polypeptide generated or polypeptide。Specifically, it is possible to use " redundancy " synthesis of genetic code or selection encode the restructuring variant of these same or similar polypeptide。Multiple codon can be introduced and replace (as formed the reticent change of multiple restriction site) with optimization clone's entrance plasmid or viral vector or the expression (namely relating to " the codon optimization " of host) in specific protokaryon or eukaryotic system。Sudden change in polynucleotide sequence can be reflected in polypeptide or be added in other peptide fragment domains of polypeptide for revising the character of polypeptide arbitrary portion, thus changing feature (such as DNA and other part binding affinities or degraded/switching rate)。According to some embodiments, the polynucleotide of the present invention are that codon is optimized, it is possible to the expression in transformed host of the optimization polypeptide。
When term " codon optimization " refers to the gene or the nucleic acid molecule encoding district that convert for multiple host, represent the codon change in gene or nucleic acid molecule encoding district, be reflected in do not change polypeptide coded by DNA when host living beings in normally used codon。This kind of optimization includes using one or more codons of more-frequently used in the gene of this biology to replace at least one or more than one or a large amount of codon。
Allow the sequence encoding this gene different including the nucleotide sequence deviation encoding the amino acid whose codon of any polypeptide chain。Owing to each codon is made up of three nucleotide, and the nucleotide containing DNA is confined to four kinds of specific base, so there being nucleotide combination 64 kinds possible, and wherein 61 kinds of coded amino acids (remaining the signal of three kinds of codon code termination translations)。Therefore, many aminoacid are specified by more than one codons。Such as, amino acid alanine and proline are encoded by 4 kinds of triplets, and serine and arginine are by 6 kinds of codings, and tryptophan and methionine are only by a kind of triplet coding。This degeneracy makes when not changing protein amino acid sequence coded by DNA, and DNA base composition can change in the broader context。
Be there is preference by many organisms in the concrete aminoacid inserted in the concrete codon encoding growth peptide chain of use。Codon preferably or the difference that between codon preference, organism, codon uses, based on the degeneracy of genetic code, and is recorded in detail in many organisms。Codon preference is often relevant with the translation efficiency of messenger RNA (mRNA), then thinks that it relies on the effectiveness etc. of codon character and concrete transfer RNA (tRNA) molecule being translated。Selected tRNA advantage in cell generally reflects codon the most used in peptide symthesis。Therefore, gene can be adjusted based on best gene expression in codon optimized just given organism。
Based on the obtainable lots of genes sequence of various animals, plant and microbial species, the relative frequency that codon uses can be calculated。Codon uses table available from such ashttp://www.kazusa.or.jp/codon/" codon uses data base " of (access on March 20th, 2008), these tables are applicable to many aspects。Referring to Nakamura etc., Nucl.AcidsRes.28:292 (2000)。
Codon is used to use table, described frequency can be applied to any given peptide sequence by those of ordinary skill in the art, and produce the nucleic acid fragment that coding region is codon optimized, described coding region coded polypeptide but use optimal codon, for expressing sequence interested in host interested。
Can manually complete with optimization frequency accidental assignment of password to encode given peptide sequence, by calculating each amino acid whose codon frequency, with backward peptide sequence random assortment codon。Additionally, those of ordinary skill in the art can be readily available many algorithms and computer software programs。Such as, " editor's sequence (EditSeq) " function in the Lasergene software kit of the DNASTAR company of state of Wisconsin Madison, retroversion (backtranslation) function in the InforMax company VectorNTI external member of Maryland State Bei Saisida, and retroversion (backtranslate) function in the Accelrys company GFG-Wisconsin software kit of San Diego, CA。Additionally, multiple resources can be used so that coding region sequence is carried out codon optimization from public channel, for instance http: " entelechon.com/bioinformatics/backtranslation.php？" retroversion (backtranslation) " function in lang=eng and " retroversion sequence (the backtranseq) " function in http " bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html "。Those of ordinary skill in the art it be also possible to use basic math function and are easily accomplished the structure of basic algorithm, with based on given frequency assignment of password。In addition, the coding region that can be optimized by multiple method pin design known in the art, described method includes software kit, such as " synthetic gene designer (syntheticgenedesigner) ", network address is http: " phenotype.biosci.umbc.edu/codon/sgd/index.php "。
In some embodiments, the present invention includes the optimized polynucleotide of codon, for expressing polypeptide interested in host interested。Therefore, for example, although the polynucleotide of the present invention both can also can be expressed at monocot plant species in dicot plant species, but still can modify to the sequence of polynucleotide to form the sub-preference of specific cryptosystem for a kind of monocotyledon interested or dicotyledon and G/C content preference。Can be readily available and use technology described herein or techniques known in the art to use codon usage table and software program routinely, any given sequence be carried out codon optimization so that it expresses in host cell interested for instructing。The sub-usage of exemplary cryptographic instructs and refers to such as Murray etc., and NucleicAcidsRes.17:477-98 (1989), the document has passed through incorporated and is incorporated herein。Polynucleotide also by the polypeptide of Recombinant design code book invention use technology known in the art can be easily accomplished identify and revise potential to subtract steady sequence and potential secondary structure to remove。
Term used herein " check plant " represents the plant of the peptide sequence without the present invention。Suitable check plant includes the non-transgenic plant in the parent system for generating transgenic plant or non-transgenic plant same kind of with transgenic plant, and this kind of plant is generally referred to as wild type herein " plant。
Term used herein " enhancing character " represents a kind of feature of transgenic plant, includes but not limited to the Agronomic character of the enhancing that the phytomorph to strengthen, physiology, g and D, yield, nutrition lifting, disease or pest resistance or environment or chemical resistance are feature。The present invention more specifically in, described enhancing character strengthens character selected from one group, including the water service efficiency (such as " drought resistance ") strengthened, the salt toleration of enhancing, the cold tolerance of enhancing, output increased and enhancing nitrogen service efficiency。
" diminishing condition " or " non-water coerce " condition is used alternatingly herein, in order to represent that plant or cell grow when not having water to limit and the major part of cell, plant or plant does not show the visible sign relevant to water deficit conditions or symptom。On the contrary, " restrict water supply condition ", " water deficit conditions " and " water stress conditions " is used alternatingly herein, in order to represent that plant or cell grow when water limits and the major part of cell, plant or plant shows visible and arid and water and coerces relevant sign or symptom。
" diminishing environment " or " non-water coerce " environment is used alternatingly herein, in order to represent that plant or cell grow when not having water to limit and the major part of cell, plant or plant does not show the visible sign relevant to water deficit conditions or symptom。On the contrary, " restrict water supply condition ", " water deficit conditions " and " water stress conditions " is used alternatingly herein, in order to represent that plant or cell grow when water limits and the major part of cell, plant or plant shows visible and arid and water and coerces relevant sign or symptom。
The implication that the implication of term used herein " output increased " is expressed when generally using with in agricultural and/or research field and understand this term is identical。The output increased of the transgenic plant of the present invention can be measured by serial of methods, including test weight, the seed number of every strain plant, seed weight, the seed number of per unit area or weight (such as every acre of seed number or seed weight)。In a detailed description of the invention, output increased represents that the seed of a strain plant or a flora (per unit area) or grain produce weight and statistically significantly increases。According to an embodiment, compared with producing weight with the seed of comparable number check plant or grain, seed or the grain weight production of the sample size (such as n=10 or more) of a statistically significant of the transgenic plant of the present invention add at least 5%。
The implication that the implication of term used herein " photosynthesis rate quickening " is expressed when generally using with in agricultural and/or research field and understand this term is identical。The photosynthesis rate of the transgenic plant of the present invention is accelerated to be measured by serial of methods, including leaf CO2Gas exchange measures。In a detailed description of the invention, photosynthesis rate accelerates to represent the leaf CO of a strain plant or a flora (per unit area)2Gas exchange statistically significantly increases。According to an embodiment, compared with the photosynthesis rate of comparable number check plant, the photosynthesis rate of the sample size (such as n=10 or more) of a statistically significant of the transgenic plant of the present invention accelerates at least 5%。
Modified HaHB4 polynucleotide and polypeptide
The present invention relates to following discovery, the transgenic plant of the polynucleotide sequence that namely can surprisingly result in the sequence modification of HaHB4 transcription factor the modified HaHB4 protein of coding inverted presents enhancing character。HaHB4 gene (include promoter and regulate sequence), protein and HaHB4 transformed host are also described in detail in U.S. Patent number 7,674,955, and the document is incorporated herein by reference of text。
In some embodiments, nucleic acid molecules provided by the present invention includes the polynucleotide sequence that encodes modified HaHB4 albumen。In other embodiment, nucleic acid molecules provided by the present invention includes coding with the polynucleotide sequence with the functional activity fragment or variant of the not homotactic modified HaHB4 of HaHB4 (SEQIDNO:2)。Present invention additionally comprises the protein coded by other polynucleotide of above-mentioned these and the present invention。
As disclosed herein, converting has the transgenic plant of modified HaHB4 (modHaHB4) protein with SEQIDNO:4 and SEQIDNO:8 aminoacid sequence to show enhancing character unexpectedly, including the yield when water is coerced with growth under non-water stress conditions higher than control group plants。Similarly, as described herein, convert have the transgenic plant of modified HaHB4 (modHaHB4) protein with SEQIDNO:4 and SEQIDNO:8 aminoacid sequence to show enhancing character unexpectedly, including water coerce with non-water stress conditions under grow time photosynthesis faster than control group plants。Therefore, in one embodiment, the nucleic acid molecules that the present invention relates to includes the polynucleotide sequence of modified Helianthi (Helianthusannuus) HB-4 (mod1HaHB4) of coding-belt SEQIDNO:4 aminoacid sequence。In a detailed description of the invention, nucleic acid includes the polynucleotide sequence of SEQIDNO:3, SEQIDNO:12, SEQIDNO:13, SEQIDNO:15 or SEQIDNO:16。In another embodiment, the nucleic acid molecules that the present invention relates to includes the polynucleotide sequence of modified Helianthi (Helianthusannuus) HB-4 (mod2HaHB4) of coding-belt SEQIDNO:8 aminoacid sequence。In a specific embodiment, nucleic acid includes the polynucleotide sequence of SEQIDNO:14 or SEQIDNO:17。
Present invention additionally comprises and convert the transgenic plant having modified HaHB4 (modHaHB4) albumen with SEQIDNO:38 and SEQIDNO:39 aminoacid sequence。In a specific embodiment, these plant performance go out to strengthen character, accelerate compared to control group plants output increased and/or photosynthesis including when water is coerced or grows under non-water stress conditions。Therefore, in one embodiment, the nucleic acid molecules that the present invention relates to includes the polynucleotide sequence of modified Helianthi (Helianthusannuus) HB-4 (mod3HaHB4) of coding-belt SEQIDNO:38 aminoacid sequence。In another embodiment, the nucleic acid molecules that the present invention relates to includes the polynucleotide sequence of modified Helianthi (Helianthusannuus) HB-4 (mod4HaHB4) of coding-belt SEQIDNO:39 aminoacid sequence。
Invention further provides the polynucleotide of the functional activity fragment of a kind of coding mod1HaHB4 (HaHB4.2 (SEQIDNO:4)), it comprises the mod1HaHB4 sequence of the corresponding sequence being not present in HaHB4 (SEQIDNO:2)。In some embodiments, described functional activity fragment comprises at least 15,20,25,30,40,50,60,75,100,125,150 or 175 continuous amino acid residues in mod1HaHB4 (SEQIDNO:4) aminoacid sequence。In other embodiments, described fragment comprises at least 10-50,25-75,50-100,75-125 or 100-175 continuous amino acid residue in mod1HaHB4 (SEQIDNO:4) aminoacid sequence。In other embodiments, described fragment includes the sequence of the amino acid residue 150-170 of amino acid residue 13-25 or SEQID:4 of amino acid residue 2-10, SEQID:4 of SEQIDNO:4。
Invention further provides the polynucleotide of the functional activity fragment of a kind of coding mod2HaHB4 (HaHb4.3 (SEQIDNO:8)), it comprises the mod2HaHB4 sequence of the corresponding sequence being not present in HaHB4 (SEQIDNO:2)。In some embodiments, described functional activity fragment comprises at least 15,20,25,30,40,50,60,75,100,125,150 or 175 continuous amino acid residues in mod2HaHB4 (SEQIDNO:8) aminoacid sequence。In other embodiments, described fragment comprises at least 10-50,25-75,50-100,75-125 or 100-175 continuous amino acid residue in mod2HaHB4 (SEQIDNO:8) aminoacid sequence。In other embodiments, described fragment comprises the sequence of the amino acid residue 2-10 of SEQIDNO:8。
Invention further provides the polynucleotide of the functional activity fragment of a kind of coding mod3HaHB4 (HaHB4.4 (SEQIDNO:38)), it comprises the mod3HaHB4 sequence of the corresponding sequence being not present in HaHB4 (SEQIDNO:2)。In some embodiments, described functional activity fragment comprises the sequence of amino acid residue 2-15 or 165-175 of SEQIDNO:38, and comprises at least 15,20,25,30,40,50,60,75,100,125,150 or 175 continuous amino acid residues in mod3HaHB4 (SEQIDNO:38) aminoacid sequence。In other embodiments, described functional activity fragment comprises the sequence of amino acid residue 2-15 or 165-175 of SEQIDNO:38, and comprises at least 10-50,25-75,50-100,75-125,100-175 continuous amino acid residue in mod3HaHB4 (SEQIDNO:38) aminoacid sequence。
Invention further provides the polynucleotide of the functional activity fragment of a kind of coding mod4HaHB4 (SEQIDNO:39), it comprises the mod4HaHB4 sequence of the corresponding sequence being not present in HaHB4 (SEQIDNO:2)。In some embodiments, described functional activity fragment comprises the sequence of the amino acid residue 2-10 of SEQIDNO:39, and comprises at least 15 in the aminoacid sequence of mod4HaHB4 (SEQIDNO:39), 20,25,30,40,50,60,75,100,125,150 or 175 continuous amino acid residues。In other embodiments, described functional activity fragment comprises the sequence of the amino acid residue 2-10 of SEQIDNO:39, and comprises at least 10-50,25-75,50-100,75-125,100-175 continuous amino acid residue in the aminoacid sequence of mod4HaHB4 (SEQIDNO:39)。
In another embodiment, the polynucleotide encoding of the present invention is equivalent to mod1HaHB4 (SEQIDNO:4) N end and/or the functional activity fragment of C end disappearance。Therefore, according to some embodiments, the functional activity fragment of the polynucleotide encoding mod1HaHB4 of the present invention, the N end of described mod1HaHB4 (SEQIDNO:4) have at least 1 but less than 5,10,15 or 20 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod1HaHB4 (SEQIDNO:4) of the present invention, the C end of described mod1HaHB4 (SEQIDNO:4) have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod1HaHB4 of the present invention, the N end of described mod1HaHB4 (SEQIDNO:4) have at least 1 but less than 5,10,15 or 20 aminoacid deletion and C end have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。
In another embodiment, the polynucleotide encoding of the present invention is equivalent to mod2HaHB4 (SEQIDNO:8) N end and/or the functional activity fragment of C end disappearance。Therefore, according to some embodiments, the functional activity fragment of the polynucleotide encoding mod2HaHB4 of the present invention, the N end of described mod2HaHB4 (SEQIDNO:8) have at least 1 but less than 5,10,15 or 20 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod2HaHB4 (SEQIDNO:8) of the present invention, the C end of described mod2HaHB4 (SEQIDNO:8) have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod2HaHB4 of the present invention, the N end of described mod2HaHB4 (SEQIDNO:8) have at least 1 but less than 5,10,15 or 20 aminoacid deletion and C end have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。
In another embodiment, the polynucleotide encoding of the present invention is equivalent to mod3HaHB4 (HaHB4.3 (SEQIDNO:38)) N end and/or the functional activity fragment of C end disappearance。Therefore, according to some embodiments, the functional activity fragment of the polynucleotide encoding mod3HaHB4 of the present invention, the N end of described mod3HaHB4 (SEQIDNO:38) have at least 1 but less than 9,10,15 or 20 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod3HaHB4 (SEQIDNO:38) of the present invention, the C end of described mod3HaHB4 (SEQIDNO:38) has at least 1 but less than 9 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod3HaHB4 of the present invention, the N end of described mod3HaHB4 (SEQIDNO:38) have at least 1 but less than 5,10,15 or 20 aminoacid deletion and C end have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。
In another embodiment, the polynucleotide encoding of the present invention is equivalent to mod4HaHB4 (SEQIDNO:39) N end and/or the functional activity fragment of C end disappearance。Therefore, according to some embodiments, the functional activity fragment of the polynucleotide encoding mod4HaHB4 of the present invention, the N end of described mod4HaHB4 (SEQIDNO:39) have at least 1 but less than 5,10,15 or 20 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod4HaHB4 (SEQIDNO:39) of the present invention, the C end of described mod4HaHB4 (SEQIDNO:39) have at least 1 but less than 10,25,50,75 or 100 aminoacid deletion。In other embodiments, the functional activity fragment of the polynucleotide encoding mod4HaHB4 of the present invention, the N end of described mod4HaHB4 (SEQIDNO:39) have at least 1 but less than 5,10,15 or 20 aminoacid deletion and C end have at least 1 but less than 7 aminoacid deletion。
The region of modHaHB4 albumen (such as, mod1HaHB11 (SEQIDNO:4) and mod2HaHB11 (SEQIDNO:8)) includes amino terminal region (" NTR ": SEQIDNO:6 or SEQIDNO:9)；Homeodomain (the amino acid residue 13-78 of " HD ": SEQIDNO:4；The amino acid residue 12-77 of SEQIDNO:8)；Leucine zipper (the amino acid residue 79-108 of " LZ ": SEQIDNO:4；The amino acid residue 78-107 of SEQIDNO:8)；And carboxy-terminal end region (the amino acid residue 109-177 of " CTR ": SEQIDNO:4；The amino acid residue 108-176 of SEQIDNO:8)。According to some embodiments, polynucleotide include the protein in the one or more regions containing mod1HaHB4 albumen, and described region is selected from following group: NTR (SEQIDNO:6 or SEQIDNO:9)；Homeodomain (the amino acid residue 12-77 of amino acid residue 13-78 or SEQIDNO:8 of SEQIDNO:4)；Leucine zipper (the amino acid residue 79-108 of SEQIDNO:4；Or the amino acid residue 78-107 of SEQIDNO:8)；And CTR (the amino acid residue 108-176 of amino acid residue 109-177 or SEQIDNO:8 of SEQIDNO:4)。Additionally providing the polynucleotide encoding two or more modHaHB4 areas combine, described region is selected from following group: NTR (SEQIDNO:6 or SEQIDNO:9)；Homeodomain (the amino acid residue 13-78 of SEQIDNO:4)；Leucine zipper (the amino acid residue 79-108 of SEQIDNO:4)；And CTR (the amino acid residue 109-177 of SEQIDNO:4)。
The region of mod3HaHB4 albumen (SEQIDNO:38) includes homeodomain (the amino acid residue 13-78 of " HD ": SEQIDNO:38)；Carboxy-terminal end region (the amino acid residue 109-177 of " CTR ": SEQIDNO:38)。According to some embodiments, polynucleotide include the protein containing the one or more region of mod3HaHB4 albumen, and described region is selected from following group: homeodomain (the amino acid residue 13-78 of SEQIDNO:38)；And CTR (the amino acid residue 109-177 of SEQIDNO:38)。
In other embodiments, the present invention includes the polynucleotide of the functional activity variant of coding mod1HaHB4 (SEQIDNO:4) or mod2HaHB4 (SEQIDNO:8), and the sequence of wherein said variant is not the sequence of HaHB4 (SEQIDNO:2)。In other embodiments, the present invention includes the polynucleotide of the functional activity variant of coding mod3HaHB4 (SEQIDNO:38) or mod4HaHB4 (SEQIDNO:39), and the sequence of wherein said variant is not the sequence of HaHB4 (SEQIDNO:2)。The variant of the present invention includes the interpolation in mod1HaHB4 or mod2HaHB4 sequence, replacement。Variant in the present invention includes the interpolation in mod3HaHB4 or mod4HaHB4 sequence, replacement。Aminoacid " replacement " refers to replace with certain monoamino-acid another structure and/or the similar aminoacid of chemical characteristic, for instance conservative amino acid is replaced。According to the similarity of the polarity of involved residue, electric charge, dissolubility, hydrophobicity, hydrophilic and/or amphiphilic nature, " conservative " aminoacid replacement can be carried out。Such as, nonpolar (hydrophobicity) aminoacid includes alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine；Polar neutral amino acid includes glycine, serine, threonine, cysteine, tyrosine, agedoite and glutamine；Positively charged (alkalescence) aminoacid includes arginine, lysine and histidine；Electronegative (acidity) aminoacid includes aspartic acid and glutamic acid。Generally " insertion " or " disappearance " range for about 1 to about 20 aminoacid, it is preferred to about 1 to about 10 aminoacid, more preferably about 2 to about 5 aminoacid。Non-conservation replacement refers to a type of member is replaced by another type of member。Such as, aminoacid replacement can also refer to replace with certain monoamino-acid another structure and/or the different aminoacid of chemical characteristic, for instance an aminoacid (such as polarity) in a group replaces with another aminoacid (such as alkalescence) of different group。Recombinant DNA technology can be used systematically the aminoacid in peptide molecule to be inserted, lack or substituted and measure the activity of the restructuring variant generated, determining variant thereby through test。
In a specific embodiment, polynucleotide encoding in the present invention functional activity variant of mod1HaHB4 or mod2HaHB4, its compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8) containing 1,2,3,4,5,10 or more replacement, insertion or disappearance, the wherein said variant corresponding aminoacid sequence without HaHB4 (SEQIDNO:2)。In some embodiments, the polynucleotide encoding functional activity variant of mod1HaHB4 or mod2HaHB4, it replaces containing 1,2,3,4,5,10 or more conservative compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8)。In other embodiments, the polynucleotide encoding functional activity variant of mod1HaHB4 or mod2HaHB4, it replaces containing 1,2,3,4,5,10 or more non-conservation compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8)。
In other embodiments, the polynucleotide encoding functional activity variant of mod1HaHB4 or mod2HaHB4 albumen, its compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8) containing 1-10,1-20 or 1-25 replacement, insert or disappearance, the wherein said variant corresponding aminoacid sequence without HaHB4 (SEQIDNO:2)。In some embodiments, the polynucleotide encoding functional activity variant of mod1HaHB4 or mod2HaHB4 albumen, it replaces containing 1-10,1-20 or 1-25 conservative compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8)。In other embodiments, the polynucleotide encoding functional activity variant of mod1HaHB4 or mod2HaHB4 albumen, it replaces containing 1-10,1-20 or 1-25 non-conservation compared with the corresponding sequence of mod1HaHB4 albumen (SEQIDNO:4) or mod2HaHB4 albumen (SEQIDNO:8)。
In a specific embodiment, the polynucleotide encoding functional activity variant of mod1HaHB4 albumen, its serine comprising the 7th inserts, the threonine of the 9th is replaced by serine, the arginine of the 18th is replaced by lysine or the lysine of the 155th replaced by phenylalanine。
In a specific embodiment, the threonine that the modified HaHB4 albumen of functional activity comprises natural HaHB4 albumen (SEQIDNO:2) the 9th is replaced by serine, the threonine of the 13rd, natural HaHB4 albumen (SEQIDNO:2) is replaced by serine, the lysine of the 22nd, natural HaHB4 albumen (SEQIDNO:2) is replaced by arginine, the phenylalanine of the 159th, natural HaHB4 albumen (SEQIDNO:2) is replaced by lysine, the leucine of the 175th, natural HaHB4 albumen (SEQIDNO:2) is replaced。
In other embodiments, the present invention includes the polynucleotide of the functional activity variant of coding mod1HaHB4 albumen, wherein said variant comprise with the aminoacid sequence at least 80% of mod1HaHB4 albumen (SEQIDNO:4), 85%, 90%, 92%, 95%, 95%, 96%, 97%, 98% or 99% identical sequence, and the corresponding aminoacid sequence that wherein said variant is without HaHB4 (SEQIDNO:2)。Additionally, the suitable polynucleotide passage with above-mentioned congener encodes containing at least 50 aminoacid, at least 100 aminoacid, at least 150 amino acid whose polypeptide, and wherein corresponding sequence is not present in HaHB4 (SEQIDNO:2)。Suitable polynucleotide passage with above-mentioned congener encodes containing at least 50 aminoacid, at least 100 aminoacid, at least 150 amino acid whose polypeptide, and the corresponding aminoacid sequence without HaHB4 (SEQIDNO:2)。
In other embodiments, the present invention includes the polynucleotide of the functional activity variant of coding mod2HaHB4 albumen, wherein said variant comprise with the aminoacid sequence at least 80% of mod2HaHB4 albumen (SEQIDNO:8), 85%, 90%, 92%, 95%, 95%, 96%, 97%, 98% or 99% identical sequence, and the corresponding aminoacid sequence that wherein said variant is without HaHB4 (SEQIDNO:2)。Suitable polynucleotide passage with above-mentioned congener encodes containing at least 50 aminoacid, at least 100 aminoacid or at least 150 amino acid whose polypeptide, and the corresponding aminoacid sequence without HaHB4 (SEQIDNO:2)。
In other embodiments, the present invention includes the polynucleotide sequence with SEQIDNO:3, SEQIDNO:12, SEQIDNO:13, SEQIDNO:15 or SEQIDNO:16 at least 80%, 85%, 90%, 92%, 95%, 95%, 96%, 97%, 98% or 99% identical polynucleotide variant, SEQIDNO:14 or SEQIDNO:17 or these sequences in the complementary strand of any one or its fragment, wherein polynucleotide sequence is not present in the corresponding polynucleotide sequence of HaHB4 (SEQIDNO:1) and does not encode HaHB4 (SEQIDNO:2)。Suitable polynucleotide passage with above-mentioned congener includes the fragment that length is at least 20 nucleotide, at least 30 nucleotide, at least 40 nucleotide, at least 50 nucleotide, at least 75 nucleotide or at least 100 nucleotide。
In another embodiment, the present invention includes a kind of nucleic acid molecules, and described nucleic acid molecules comprises the polynucleotide sequence encoding following albumen: (a) mod1HaHB4 (SEQIDNO:4)；B the functional activity fragment of () described modified albumen, the aminoacid sequence of wherein said fragment is not present in HaHB4 (SEQIDNO:2)；Or the functional activity variant of (c) modified HaHB4 albumen, it comprises the aminoacid sequence with SEQIDNO:4 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny；The aminoacid sequence of wherein said fragment or variant is not present in the aminoacid sequence of HaHB4 (SEQIDNO:2)。
In another embodiment, the present invention includes a kind of nucleic acid molecules, and described nucleic acid molecules comprises the polynucleotide sequence encoding following albumen: (a) mod1HaHB4 (SEQIDNO:4)；B the functional activity fragment of () mod1HaHB4, the aminoacid sequence of wherein said fragment is not present in HaHB4 (SEQIDNO:2)；Or the functional activity variant of (c) mod1HaHB4 albumen, wherein said variant comprises the aminoacid sequence with SEQIDNO:4 and at least there is the aminoacid sequence of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeny, and the aminoacid sequence of wherein said fragment or variant is not present in HaHB4 (SEQIDNO:2)。
Invention further provides the nucleic acid (including encoding the modHaHB4 fragment of polynucleotide described herein) comprising a kind of polynucleotide sequence, its nucleic acid hybridization under stringent hybridization condition and containing coding mod1HaHB4 (SEQIDNO:4) or the polynucleotide complementary strand of mod2HaHB4 (SEQIDNO:8), the polynucleotide encoding one wherein hybridized is different from the functional activity polypeptide of HaHB4 (SEQIDNO:2)。In other embodiments, the nucleic acid hybridization of polynucleotide polynucleotide sequence complementary strand under strict hybridization conditions and containing SEQIDNO:3 or SEQIDNO:14, wherein hybrid polynucleotide coding one is different from the functional activity polypeptide of HaHB4 (SEQIDNO:2)。
In this article, when the polynucleotide of single stranded form or nucleic acid fragment can be annealed with other nucleic acid fragment when suitable temperature and solution ion strength, then claiming these polynucleotide is " interfertile " with other polynucleotide or nucleic acid fragment (such as cDNA, genomic DNA or RNA molecule)。Hybridization is well-known with wash conditions and is illustrated in Sambrook, J., Fritsch, and Maniatis E.F., T.MolecularCloning:ALaboratoryManual (" molecular cloning: laboratory manual "), the second edition, cold spring gulf laboratory Press (ColdSpringHarborLaboratory): cold spring port, New York (1989), is specially Chapter 11 therein and table 11.1 (document is incorporated herein by reference of text)。Temperature and ionic strength conditions determine " preciseness " of hybridization。Adjustable stringent conditions is to screen medium similar fragment (homologous sequence as from sibship organism farther out), to highly similar fragment (as making the gene of copy function enzyme from the nearlyer organism of sibship)。Washing after hybridization determines high stringency conditions。One set condition uses a series of washing, starts from 6XSSC, 0.5%SDS room temperature 15 minutes, then uses 2XSSC, 0.5%SDS45 DEG C of repeated washing 30 minutes, then washs repetition in 30 minutes twice with 0.2XSSC, 0.5%SDS50 DEG C。Another group high stringency conditions uses higher temperature, wherein washs ibid, except last twice is increased to 60 DEG C by 0.2XSSC, 0.5%SDS washing temperature of 30 minutes。And another organize high high stringency conditions uses twice finally washing be 0.1XSSC, 0.1%SDS, 65 DEG C。Another is organized high high stringency conditions and such as includes in 0.1XSSC, 0.1%SDS, 65 DEG C of hybridization and wash with 2XSSC, 0.1%SDS then 0.1XSSC, 0.1%SDS。
Hybridization needs two nucleic acid comprising complementary series, although the preciseness according to described hybridization, it is possible to there is the mispairing between base。The appropriate stringency of hybrid nucleic acid depends on the length of nucleic acid and complementary degree, and this is variable well known in the art。Between two nucleotide sequences, the degree of similarity or homology is more high, has the T of the nucleic acid hybrids of these sequencesmIt is worth more high。The relative stability (Tm corresponding to higher) of nucleic acid hybridization weakens in the following order successively: RNA:RNA, DNA:RNA, DNA:DNA。For the length crossbred more than 100 nucleotide, derive the equation (see Sambrook etc., ibid, 9.50-9.51) calculating Tm。Hybridization for shorter nucleic acid (such as oligonucleotide), the position of mispairing becomes more important, and the length of oligonucleotide determines its specificity (see Sambrook etc., ibid, 11.7-11.8)。In one embodiment, the length of hybrid nucleic acid 10 nucleotide can be at least about。In another embodiment, the shortest length of hybrid nucleic acid 15 nucleotide can be at least about。And in another embodiment, the shortest length of hybrid nucleic acid 20 nucleotide or at least about 30 nucleotide can be at least about。It addition, those skilled in the art will readily recognize that, according to factors such as such as probe length, it may be necessary to adjust temperature and washing liquid salinity。
Standard recombinant dna used herein and molecule clone technology are well known in the art, and it is described in Sambrook, J., Fritsch, and Maniatis E.F., T., MolecularCloning:ALaboratoryManual (" molecular cloning: laboratory manual "), the second edition, cold spring gulf laboratory Press (ColdSpringHarborLaboratoryPress), cold spring port, New York (1989) (hereinafter referred to as " Maniatis ")；And Silhavy, T.J., Bennan, and Enquist M.L., L.W., ExperimentswithGeneFusions (" gene fusion experiments "), cold spring gulf laboratory Press (ColdSpringHarborLaboratoryPress), cold spring port, New York (1984)；And Ausubel, F.M. etc., (" molecular biology experiment guide ", Green Publishing Group and power publishing company (GreenePublish.Assoc.&WileyInterscience) publish (1987) to CurrentProtocolsinMolecularBiology；Frohman (Frohman clone PCR product。In polymerase chain reaction, K.B.Mullis, F.Fre and R.A.Gibas edit, 14-37 page；Every section of document is incorporated herein each through incorporated), for generating the albumen with different aminoacids sequence, for instance produce amino acid whose replacement, disappearance and/or insertion。
Carrier and host cell
Present invention additionally comprises the carrier containing modHaHB4 polynucleotide (including expression cassette)。
The carrier of the present invention can be made up of DNA or RNA, it is possible to is wire or closed hoop plasmid。Described carrier can be clone, expand, shuttle back and forth or expression vector。Carrier system can be single carrier or plasmid or two or more carrier jointly comprised or control duplication, integration and/or the expression in host cell of the polynucleotide of the present invention。According to any specific promoter sequence contained in carrier, it is possible to orientation forward or backwards by the polynucleotide insertion vector of the present invention。
Carrier component for transformation of host cells (such as antibacterial or plant) typically includes, but not limited to following one or more: signal sequence, replication origin, one or more selected marker and optional promoter (such as inducible promoter), is used for making exogenous DNA express。After being typically chosen property marker gene coding one converts, host cell is survived in selective medium or grows necessary protein。Typical Select gene coding gives the albumen to antibiotic or other toxin (such as ampicillin, kanamycin, tetracycline, neomycin, trimethoprim, spectinomycin, sulfonamide or methotrexate) resistance, (b) extra-nutrition deficiency defect, or the critical nutrients that (c) supply cannot obtain from complicated culture medium。These successful conversion Hemapoiesis one albumen of heterologous protein or its fragment, described albumen gives the cell resistance to medicine, thus surviving in selection scheme。
Recombinant DNA technology by standard, it is possible to build the suitable carrier of the polynucleotide comprising one or more component listed above and the present invention, and method available in this area and reagent can be used easily to prepare。Referring to such as Sambrook etc., ibid and Ausubel etc., ibid。Generally, can conventional cutting, adjust and reconnect separation after nucleic acid plasmid or the DNA fragmentation vector construction that comprises required related component with formation, thus giving function and the characteristic that this structure needs。Multiple suitable carrier and promoter commercially and/or are as known in the art, can carry out routinely according to the present invention using or modifying。The representative illustration of bacteria carrier includes: pQE70, pQE80L, pQE81L, pQE82L, pQE60 and pQE-9 (Kai Jie company (Qiagen))；PBS, pD10, phagescript, psiX174, pBS.RTM.SK, pBS.RTM.KS, pNH8A, pNH16a, pNH18A, pNH46A；PGEX-3X, pGEX-4T-1 to pGEX-4T-3, pGEX-5X-1 to pGEX-5X-3 and pGEX-6P-1 to pGEX-6P-3；PBluescriptSK and pBluescriptKS and pBluescriptII (Si Cha column foot company (Stratagene), La Youla city, California), pIN carrier (VanHeeke and Schuster, 1989), pTRC99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia biotechnology (Pharmacia), Uppsala, SWE)。The representative illustration of eukaryotic vector includes: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Si Cha column foot company (Stratagene)), pSG5, pSVK3, pBPV, pMSG, pSVL, GEM1 (Pu Luomaige biotech company (PromegaBiotec), state of Wisconsin Madison) and pSVLSV40。
In some embodiments, the polynucleotide of the present invention are inserted in carrier, are operably connected in host plant function with suitable promoter, drive the expression of polynucleotide sequence。It is it known in the art, to select for building and using according to the present invention based on some factors routinely suitable in many carriers of this purpose, the particular host cell that described factor includes the size of the polynucleotide sequence of such as insertion vector and carrier converts。The plant conversion carrier of the present invention also contains one section of HaHB4 having function or heterologous intron sequences in coded sequence upstream or the coded sequence interior location even at the polynucleotide of the present invention, also can contain one section of 5 ' end (5 ') untranslated leader (i.e. UTR or 5'-UTR) in position between promoter and translation starting point。
The carrier of the present invention preferably comprises a selected marker, and described labelling gives selectivity phenotype, it is possible to identify the cell of the polynucleotide expressed after converting and/or containing the present invention。Such as, in multiple embodiments, selected marker encodes a kind of albumen, gives Biocide resistance, antibiotic resistance (such as kanamycin, G418, bleomycin, hygromycin etc.) or Herbicid resistant (such as glyphosate etc.)。Coding kalamycin resistance nptII gene (Messing etc., the Gene19:259-268 (1982) of the neo gene that kanamycin can be used to select, imparting kanamycin and associated antibiotic resistance can be included but not limited to according to the example of the selected marker that the compositions and methods of the invention use；Bevan etc., Nature304:184-187 (1983)), the bar gene (White etc. of conferring herbicide phosphine oxamate resistance, Nucl.AcidsRes.18:1062 (1990), Spencer etc., Theor.Appl.Genet.79:625-631 (1990)), give hph gene (Blochinger etc., Mol.Cell.Biol.4:2929-2931 (1984)) to antibiotic hygromycin resistance；G418, the mutant EPSP synthase gene (U.S. Patent number 4,940,835 and 5,188,642) of coding glyphosate resistance；Give the mutant acetolactate synthase gene (ALS) of imidazolone or sulphur urea resistance；Give the nitrilase gene of brdmo iltesi；With methotrexate resistance DHFR gene。Additionally, multiple choices labelling can be used to give ampicillin, bleomycin, chloromycetin, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphine oxamate, puromycin, spectinomycin, rifampicin and tetracycline。The example of this kind of selected marker is listed in U.S. Patent number 5,550,318；5,633,435；5,780,708 and 6,118,047, the full content of above-mentioned each patent is incorporated herein each through incorporated。
According to some embodiments, the present invention relates to nucleic acid (including carrier and expression cassette), described nucleic acid comprises the polynucleotide of the present invention being operably connected with promoter。In some embodiments, the polynucleotide of the present invention are operably connected with constitutive promoter。In a specific embodiment, described constitutive promoter is 35SCaMV promoter or Ubi promoter。According to other embodiments, the polynucleotide of the present invention are operably connected with inducible promoter。In a specific embodiment, described inducible promoter is stress induced promoter。In other embodiments, described inducible promoter is the modified HaHB4 promoter merged with the First Intron of arabidopsis Cox2-c。
In other embodiments, the carrier of the present invention comprises that HaHB4 little allele promoter sequence (SEQIDNO:20), HaHB4 big allele promoter sequence (SEQIDNO:21) or HaHB4 promoter be big or the functional activity fragment of little allele promoter sequence or derivant。According to an embodiment, described promoter comprises the 805th to 1221,904 to 1221,1011 to 1221 or 15 to 622 nucleotide sequences of the little allele promoter sequence (SEQIDNO:20) of HaHB4。According to another embodiment, described promoter comprises 15-409 or 805 to 1221 nucleotide sequence of the big allele promoter sequence (SEQIDNO:21) of HaHB4。According to another embodiment, described promoter comprises 15-409 or 805 to 1221 nucleotide sequence of the big allele promoter sequence (SEQIDNO:21) of HaHB4。In another embodiment, described promoter comprises the fragment sequence of at least 50,100,150,200,250,300,350,400,450,500,600,700 or 800 nucleotide of the little allele promoter sequence (SEQIDNO:20) of HaHB4。In another embodiment, described promoter comprises the fragment sequence of at least 50,100,150,200,250,300,350,400,450,500,600,700 or 800 nucleotide of the big allele promoter sequence (SEQIDNO:21) of HaHB4。
Other promoteres multiple being active in plant cell are to it known in the art, and can provide in plant expression vector according to multiple embodiments of the present invention。These promoteres include the promoter that exists in Plant Genome and the promoter from virus, antibacterial and other sources, including rouge alkali synthetase (NOS) promoter carried on the tl plasmid of Agrobacterium and octopine synthase (OCS) promoter and Cauliflower Mosaic viroid promoter (such as cauliflower mosaic virus or figwort mosaic virus promoter)。Referring to the U.S. Patent number 5,858,742 and 5,322,938 such as disclosing the constitutive promoter deriving from cauliflower mosaic virus (CaMV35S), disclose the U.S. Patent number 5,378,619 of figwort mosaic virus (FMV) 35S promoter, disclose the U.S. Patent number 6,437,217 of Semen Maydis RS81 promoter, disclose the U.S. Patent number 5,641,876 of rice actin promoters, disclose the U.S. Patent number 6,426,446 of Semen Maydis RS324 promoter, disclose the U.S. Patent number 6,429,362 of Semen Maydis PR-1 promoter, disclose the U.S. Patent number 6,232,526 of Semen Maydis A3 promoter, disclose the U.S. Patent number 6,177,611 of composing type corn promoter, disclose the U.S. Patent number 6,433,252 of Semen Maydis L3 oleosin promoter, disclose the U.S. Patent number 6,429,357 of rice actin 2 promoter and intron, disclose the U.S. Patent number 5,837,848 of root-specific promoter, disclose the U.S. Patent number 6,084,089 of cold inducible promoter, disclose the U.S. Patent number 6,294,714 of Light-inducible promoter, disclose the U.S. Patent number 6,140,078 of Salt treatment type promoter, disclose the U.S. Patent number 6,252,138 of pathogen-inducible promoter, disclose phosphorus and lack the U.S. Patent number 6,175,060 of inducible promoter, disclose the 5' for designing effective plant expression vector, the U.S. Application Publication No 2002/0192813A1 of 3' and intron element, disclose the U.S. Application Publication No 09/078,972 of a kind of coixin promoter, disclosing the U.S. Application Publication No 09/757,089 of a kind of DCIPThe chloroplast of maize aldolase promoter and disclose the U.S. Application Publication No 10/739,565 of water defect inducible promoter, above-mentioned document is incorporated herein each through quoting。These and other promoteres of function in plant cell multiple be it known in the art, and can be operably connected with the polynucleotide of the present invention and/or in the carrier of the present invention to drive or to control the expression of polynucleotide sequence in transgenic plant cells。
In other embodiments, the polynucleotide (individually or with carrier sequence together) of the present invention are operably connected with the promoter in the regulation and control region deriving from the plant gene of process LAN under water deficit conditions。In a specific embodiment, the polynucleotide of the present invention are operably connected with the promoter deriving from corresponding a certain gene 5 ' regulation and control region, and described gene is selected from: heatshock protein 17.5 (HSP17.5), HVA22 (HVA22), Rab17 or cinnamic acid 4-hydroxylase (CA4H)。Exemplary water deficit-inducible promoters disclosed in U.S. Application Publication No 10/739,565, it is incorporated herein by reference of text。
In other embodiments, the polynucleotide of the present invention are operatively connected with a kind of promoter, and and then be operably connected with one or more " enhancer sequence ", described enhancer can promote the gene expression that this promoter drives。Generally orientation forward or backwards this kind of enhancer can be inserted 5' or the 3' position of coded sequence, it is also possible to be present in intron。Multiple enhancer is known and/or reagent known in the art or technology can be used easily to identify。The exemplary enhancer can being operably connected with the polynucleotide of the present invention includes the 5' intron of rice actin 1 and rice actin 2 gene and from the element (Odell etc. of CaMV35S promoter, Nature6:810-812 (1985)), octopine synthase genes, maize alcohol dehydrogenase gene, Semen Maydis shrinks 1 (shrunken1) gene, Adh introne 1 (Callis etc., GenesandDevelop., 1:1183 (1987)), sucrose synthase intron (Vasil etc., PlantPhysiol.91:1575 (1989)) and TMVomega element (Gallie etc., PlantCell1:301 (1989))。
In other embodiments, the polynucleotide of the present invention (individually or with carrier sequence together) are operably connected with transcription terminator, the polynucleotide that described transcription terminator is responsible for terminating exceeding the present invention transcribe and correct mRNA polyadenylation。The example of the known suitable transcription terminator having function in plant includes but not limited to CaMV35S terminator, tml terminator, derives from the nopaline syntase terminator (Bevan etc. of Agrobacterium, Nucl.AcidsRes., 11:369 (1983), Depicker etc., J.Mol.Appl.Genet.1:561-573 (1982)), pearbcSE9 terminator, the octopine synthase genes from Agrobacterium and the protease inhibitor I from Rhizoma Solani tuber osi or Fructus Lycopersici esculenti or II gene 3 ' the T7 transcript terminators held。
Preferred plant conversion carrier includes carrier (such as U.S. Patent number 5,981,840,5 deriving from Agrobacterium Ti plasmid, 501,967,4,536,475,4,658,082,4,693,977 and 4,886, described in 937, and Simpson etc., PlantMol.Biol.6:403-15 (1986) and EP0122791), the content of above-mentioned each document is incorporated herein each through incorporated。Other preferred plant conversion carriers include such as Herrera-Estrella etc., Nature303:209-213 (1983)；Carrier disclosed in Bevan, M., Nucl.AcidsRes.12:8711-8721 (1984) and EP0120516, the content of above-mentioned each document is incorporated herein each through incorporated。For the plant conversion system based on Agrobacterium, other elements that conversion carrier exists in building include T-DNA left and right border sequence, promote that recombination of polynucleotide is integrated in Plant Genome。Description for the agrobacterium vector system and method for Agrobacterium-mediated gene transfer can referring to such as Gruber etc., " VectorsforPlantTransformation " (" carrier for Plant Transformation "), method in molecular biology of plants and biotechnology, ibid and Moloney etc., PlantCellReports8:238 (1989)。According to other embodiments, described carrier is a part for Binary vector systems, such as pBin19, pC22, pGA482, pCV001, pJJ1881, pPZP111, pPVP, pGreen0029, pCGN1547, pMON10098, pBI121 (Bevan, Nucl.AcidsRes.12:8711-8721 (1984), pBI101 (Jefferson etc., EMBOJ.6:3901-3907 (1987)) Risch etc., PlantMol.Biol.27:405-409 (1995), and Rothstein etc., Gene53:153-161 (1987), also can referring to Becker etc., PlantMol.Biol.20:1195-1197 (1992) and Hajdukiewicz etc., PlantMol.Biol.25:989-994 (1994), above-mentioned each document is incorporated herein each through incorporated)。
Other carriers (include expression cassette) and extracorporeal culturing method and reagent for plant cell or metaplasia and plant regeneration is to it known in the art, and can easily apply or revise for the present invention。Manufacture and use the necessary clone of polynucleotide, polypeptide, host cell and transgenic plant in the present invention, nucleic acid to handle and be commonly known in the art with synthesis, vector construction and other recombinant techniques, such as can referring to Sambrook etc., MolecularCloning:ALaboratoryManual (" molecular cloning: laboratory manual "), the second edition, cold spring port, New York, CSH Press (ColdSpringHarborLaboratoryPress) (1989)；And Ausubel etc., CurrentProtocolsinMolecularBiology (" molecular biology experiment guide "), John Wei Lisen publishing house (john wiley & sons), New York (1989)；And Kriegler, GeneTransferandExpression:ALaboratoryManual (" gene transfer and expression: laboratory manual ") (1990), it is incorporated herein by reference of text。
Generally preferable non-specific sites in Plant Genome introduces functional recombinant DNA。In particular situations, it is possible to adopt and insert recombinant DNA construction by site-specific integration。Known several site-specific recombination systems having function in plant include cre-lox disclosed in such as U.S. Patent number 4,959,317 and FLP-FRT disclosed in U.S. Patent number 5,527,695, and the content of these two sections of documents is incorporated herein each through incorporated。
Present invention also offers the host cell of carrier (including expression cassette) and/or the polynucleotide comprising the present invention。The host cell of the polynucleotide comprising the present invention includes but not limited to antibacterial (such as escherichia coli), fungus, insecticide, plant and animal cell。The polynucleotide of the present invention can be integrated in host cell gene group or make it be present in host chromosome outer (autonomously replicating plasmid as with a replication origin)。It is operably connected according to some embodiments, the polynucleotide sequence of the present invention and a promoter。
Proper method for transformation of host cells and suitable in the present invention be believed to comprise known in the art, can by the almost any means DNA (instantaneous or stably) transfered cell。Such as, the polynucleotide of the present invention can be converted routinely to bacterial host cell, as converted to escherichia coli and other hosts (Davis etc., BasicMethodsinMolecularBiology (" molecular biological basic methods ") (1986)) by calcium phosphate transfection, the transfection of DEAE-glucosan mediation or the mode of electroporation。
Or, other technologies can be used (as used CaCl2DNA is directly taken in protoplast by precipitation, polyvinyl alcohol or poly-L-Orn) polynucleotide of the present invention are converted to host cell, (referring to such as Hain etc., Mol.Gen.Genet.199:161 (1985)；With Draper etc., PlantCellPhysiol.23:451 (1982))。
In other embodiments, employ a technology as known in the art to be converted to host cell by the polynucleotide of the present invention。The example technique of transformed host cell includes protoplast transformation (referring to such as U.S. Patent number 5,508,184), microinjection (Crossway etc., Biotechniques4:320-334 (1986)；With U.S. Patent number 6,300,543), electroporation (Riggs etc., Proc.Natl.Acad.Sci.USA83:5602-5606 (1986), Fromm etc., Proc.Natl.Acad.Sci.USA82:5824-5828 (1985)), direct gene transfer (Paszkowski etc., EMBOJ.3:2717-2722 (1984)), ultrasonic method (Bao etc., UltrasoundinMedicine&Biology23:953-959 (1997)；Finer etc., Lett.Appl.Microbiol.30:406-10 (2000)；Amoah etc., J.Exp.Bot.52:1135-42 (2001))；Polyethylene glycol method (Krens etc., Nature296:72-77 (1982))；The DNA of dehydration/suppression mediation absorbs (Potrykus etc., Molec.Genet.199:183 (1985)), electroporation (U.S. Patent number 5,384,253), silicon carbide fibre stirring (U.S. Patent number 5,302,523 and 5,464,765), Agrobacterium-medialed transformation (U.S. Patent number 5,563,055；5,591,616；5,693,512；5,824,877；5,981,840；6,384,301) and DNA coated microparticle bombardment (U.S. Patent number 6,399,861；6,160,208；6,403,865；5,015,580；5,550,318；5,538,880；4,945,050；International Publication No. WO91/10725；And McCabe etc., Biotechnology6:923-926 (1988)), Sanford, Physiol.Plant79:206 (1990)；And Klein etc., Biotechnology10:268 (1992)), Tomes etc., PlantCell, TissueandorganCulture, FundamentalMethods (" plant cell, tissue and organ culture, basic methods ") in 197-213 page " DirectDNATransferintoIntactPlantCellsViaMicroprojectileB ombardment " (by the complete plant cell that DNA is directly shifted by microparticle bombardment), O.L.Gamborg and G.C.Phillips edits, Springer Fu Laige publishing house (Springer-Verlag), Heidelberg, Berlin, New York, 1995；Padgette etc., 1995)。
For many cells species, technology render transgenic cell known in the art can being used to live again as genetically modified organism, described technology is described in Christou etc., PlantPhysiol.87:671-674 (1988) (Semen sojae atricolor)；Datta etc., Biotechnology8:736-740 (1990) (Oryza sativa L.)；Klein etc., Proc.Natl.Acad.Sci.USA85:4305-4309 (1988) (Semen Maydis)；Klein etc., Biotechnology6:559-563 (1988) (Semen Maydis)；International Publication No. WO91/10725 (Semen Maydis)；Klein etc., PlantPhysiol.91:440-444 (1988) (Semen Maydis)；With Gordon-Kamm etc., PlantCell2:603-618 (1990) (Semen Maydis)。
Generally can use and be generally used for one or more technology of directly transmitting to cell the polynucleotide of the present invention (instantaneous or stably) are imported in plant。This kind of method can be varied from according to the difference of vegetation type (such as unifacial leaf or dicotyledonous) and the plant part carrying out genetic modification。The method of multiple use recombinant DNA formation plant nucleolus is known and can be used according to the invention。Two kinds of normally used methods for plant transformation are Agrobacterium-medialed transformation and microparticle bombardment。Exemplary microparticles blast technique is disclosed in U.S. Patent number 5,015,580 (Semen sojae atricolor)；5,550,318 (Semen Maydiss)；5,538,880 (Semen Maydiss)；5,914,451 (Semen sojae atricolor)；6,160,208 (Semen Maydiss)；6,399,861 (Semen Maydiss) and 6,153,812 (Semen Tritici aestivis)。Exemplary Agrobacterium-medialed transformation method is described in U.S. Patent number 5,159,135 (Cotton Gossypii)；5,824,877 (Semen sojae atricolor)；5,591,616 (Semen Maydiss)；With 6,384,301 (Semen sojae atricolor), and Horsch etc., Science227:1229-31 (1985), the content of above-mentioned document is incorporated herein each through incorporated。In other embodiments, Agrobacterium-medialed transformation is according to Hofgen etc., method disclosed in NucleicAcidResearch16:9977 (1998) or use infusion process (flower-dipping method) (such as Clough etc., described in PlantJ.16:735-743 (1998)) carry out, the content of above-mentioned document is incorporated herein each through incorporated。
The plant cell recipient of the polynucleotide of the present invention includes but not limited to plant cell culturing liquid, meristematic cell, callus, jejune embryo and pollen and ovum。According to some embodiments, host plant cell is dicotyledonous plant cells。According to other embodiments, host plant cell is monocot plant cell。Once complete to convert, namely plant cell can be used for regenerating plants。Exemplary conversion reagent and method for preparing transgenic plant are described in such as U.S. Patent number 6,194,636,6,232,526 and 4, and 658,082 and Shahin, Theor.Appl.Genet.69:235-40 (1985)；The content of above-mentioned document is incorporated herein each through incorporated。
The present invention includes the polynucleotide containing the present invention or the transgenic plant seed of polypeptide, pollen and plant part。In some embodiments, the polynucleotide of the present invention are integrated in transgenic plant DNA。In certain embodiments, the polynucleotide stable integration of the present invention is to host genome DNA。In other embodiments, the polynucleotide of the present invention are present in chromosome outer (such as with the autonomously replicating plasmid of a transcripting start point) in plant host cell。It is operably connected according to some embodiments, the polynucleotide of the present invention and a promoter。In other embodiments, at least one cell in transgenic plant, plant cell, seed, pollen or plant part expresses the polypeptide that maybe can express the present invention。And in another embodiment, at least one cell in transgenic plant, plant cell, seed, pollen or plant part expresses a kind of albumen that maybe can express the present invention, and described albumen can in vitro in conjunction with the endogenous noncoding DNA sequence of host cell。
" host " plant, plant cell, seed, pollen and the plant part (and its sexual or monogony offspring) that can use in the method in accordance with the invention and generate include the polynucleotide of the present invention can (instantaneous or stably) substantially any plant species of importing。The host plant of the present invention and host plant cell include crop plants or for producing the plant of food or feedstuff。According to some embodiments, host plant, plant cell, seed, pollen and plant part are monocotyledons。In some embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Semen sojae atricolor (Glycinemax)。In some embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Semen Tritici aestivi (Triticumaestivum)。In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Semen Maydis (Zeamays)。In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Oryza sativa L. (Oryzasativa)。In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Cotton Gossypii (Gossypiumbarbadense, Gossypiumhirsutum)。In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are Caulis Sacchari sinensis (Saccharumspp.)。In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are arabidopsis (such as Arabidopsisthaliana)。
According to other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are selected from following group: Herba Medicaginis (Medicagosativa), Brassica campestris L is (such as B.napus, B.rapa, B.juncea): especially as the rape variety (including Semen Brassicae Campestris) in seed oil source, Helianthi (Helianthusannuus), Flos Carthami (Carthamustinctorius), Semen arachidis hypogaeae (Arachishypogaea), Sorghum vulgare Pers. (Sorghumbicolor, Sorghumvulgare), Herba bromi japonici, rye (Secale cereale L.) (Secalecereale), foxtail millet is (such as pearl millet (Pennisetumglaucum), broomcorn millet (Panicummiliaceum), Semen setariae (Setariaitalica), ragimillet (Eleusinecoracana), Nicotiana tabacum L. (Nicotianatabacum), Fructus Hordei Vulgaris (Hordeum), Herba bromi japonici (Avenasativa), Fructus Lycopersici esculenti (Lycopersiconesculentum), Fructus Cucurbitae moschatae, melon (such as Fructus Melo (C.melon) and hami melon (C.cantalupensis)), Caulis Sacchari sinensis (Saccharumspp.), the legume crop of non-Semen sojae atricolor, and amyloid tuber and tuber, such as Rhizoma Solani tuber osi (Solanumtuberosum), sweet Rhizoma Solani tuber osi (Ipomoeabatatus), Maninot esculenta crantz. (Manihotesculenta), Fructus Colocasiae Esculentae, Canna generalis Bailey and Radix Betae (Betavulgaris)。
In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are vegetables。The vegetable host cell (plant) of the present invention includes such as Caulis et Folium Lactucae sativae (such as Lactucasativa), Semen phaseoli radiati (Phaseolusvulgaris), Kidney bean (Phaseoluslimensis), Semen Pisi sativi (Lathyrusspp.) and Cucumis member (such as Fructus Cucumidis sativi (C.sativus))。
In other embodiments, host plant, plant cell, seed, pollen, plant part or its transgenic product are selected from following group: coffee (Cofeaspp.), Cortex cocois radicis (Cocosnucifera), Fructus Ananadis comosi (Ananascomosus), mandarin tree (Citrusspp.), cocoa (Theobromacacao), tea (Camelliasinensis), Fructus Musae (Musaspp.), avocado (Perseaameericana), Fructus Fici (Ficuscasica), Fructus psidii guajavae immaturus (Psidiumguajava), Fructus Mangifera Indicae (Mangijeraindica), Fructus Canarii albi (Oleaeuropaea), Fructus Chaenomelis (Caricapapaya), Fructus anacardii (Anacardiumoccidentale), macadimia nut (Macadamiaintegrifolia) and almond (Prunusamygdalus)。
The cell of seed, pollen, tissue, cell and transgenic plant offspring and the present invention is also within the scope of the invention。
In one embodiment, the present invention includes the transgenic plant using a kind of nucleic acid molecules to convert, described nucleic acid molecules comprises the polynucleotide sequence of the present invention, wherein polynucleotide sequence expresses to generate recombiant protein in plant, and under the same conditions, recombiant protein makes the yield wild-type variety higher than this plant of plant。According to some embodiments, described plant is monocotyledon。In another embodiment, described plant is Semen Maydis。In another embodiment, described plant is Semen Tritici aestivi。In another embodiment, described plant is Oryza sativa L.。According to other embodiments, described transgenic plant is dicotyledon。In one embodiment, described transgenic plant is Semen sojae atricolor。In another embodiment, described transgenic plant is arabidopsis。
In another embodiment, the present invention includes the transgenic plant seed using a kind of nucleic acid molecules to convert, described nucleic acid molecules comprises the polynucleotide sequence of the present invention, wherein polynucleotide sequence expresses to generate recombiant protein in plant seed, and under the same conditions, described recombiant protein makes plant that the resistance of arid is better than the wild-type variety of this plant。According to some embodiments, described transgenic plant seed is monocotyledon。In one embodiment, described transgenic plant seed is Semen Maydis。In one embodiment, described transgenic plant seed is Semen Tritici aestivi。In another embodiment, described transgenic plant seed is Oryza sativa L.。According to other embodiments, described transgenic plant seed is dicotyledon。In one embodiment, described transgenic plant seed is Semen sojae atricolor。In another embodiment, described transgenic plant seed is arabidopsis。
In another embodiment, the present invention includes a kind of method producing high-yield transgenic host, described method includes (a) and uses the nucleic acid molecules of the polynucleotide sequence comprising the present invention stably to convert plant cell, wherein said nucleic acid molecules can be expressed in plant cell, and described cell regeneration is plant by (b)。According to an embodiment, described plant cell is monocotyledon。In another embodiment, described plant cell is Semen Maydis。In one embodiment, plant cell is Semen Tritici aestivi。In another embodiment, described plant cell is Oryza sativa L.。According to other embodiments, described plant cell is dicotyledon。In one embodiment, described plant cell is Semen sojae atricolor。In another embodiment, described plant cell is arabidopsis。
It is it known in the art, and be varied from according to the difference of plant species from the method for transgenic cell aftergrowth and plant tissue and reagent。Generally, Growth of Cells forms callus, and from wound healing, induced synthesis bud tissue, takes root subsequently。Or, can in callus induced synthesis embryo。These embryos sprout as native embryo, form plant。Culture medium usually contains enough components maintaining Growth of Cells and division, and includes such as aminoacid and hormone (such as auxin and cytokine) to maintain growth Cell differentiation inducing activity。
Predict that the polynucleotide stable integration in the present invention is to after in host genome DNA, it is possible to by the mode of sexual hybridization, these polynucleotide are transferred to other plant。Can using any number of standard breeding techniques, this depends on treating hybrid species。Once form such transgenic plant, can planting plants according to conventional methods so that nucleic acid construct is present in obtained plant。Or, transgenic seed or brood body (such as cutting) can be reclaimed from transgenic plant。Can these seed be implanted in soil subsequently and use conventional method to cultivate to form transgenic plant。
In some embodiments, the method that the invention provides plantation transgenic plant, the method includes (a) plantation and includes the transgenic seed of the nucleic acid of the present invention, carrier and/or expression cassette and (b) from this transgenic seed growth transgenosis plant。In some embodiments, the method also includes the step of results transgenic plant。In other embodiment, the method also includes the step replanting the seed from transgenic plant。In some embodiments, described transgenic plant is monocotyledon。In other embodiment, described transgenic plant is Semen Maydis。In other embodiments, described transgenic plant is Semen Tritici aestivi。In some embodiments, described transgenic plant is Oryza sativa L.。In other embodiments, described transgenic plant is dicotyledon。In some embodiments, described transgenic plant is Semen sojae atricolor。
Using the transgenic plant that Agrobacterium-mediated Transformation method is formed to generally comprise single simple recombinant DNA sequence, it inserts in chromatin, is referred to as a transgenic event。This kind of transgenic plant can be called the heterozygote of inserted exogenous sequence。By making the transgenic plant (such as F0 is for plant) of the independent isolating containing single allogenic gene sequence generate F1 generation seed with self carrying out sexual cross (" selfing "), the transgenic plant homozygote about a kind of transgenic can be obtained。In the F1 generation seed generated 1/4th are the homozygotes about this kind of transgenic。Sprout F1 generation seed and can form the plant for testing heterozygosity, it is common to use heterozygote can be distinguished and homozygous SNP measures or hot amplification assay (i.e. zygosity determination)。One strain heterozygote plant and self or another strain heterozygote plant hybridization can be produced heterozygote offspring, and homozygous transgenic offspring and the blank offspring isozygotied。
Polynucleotide except with the present invention directly convert outside plant, also by by the second strain plant hybridization of the first strain plant of the polynucleotide with the present invention and polynucleotide without the present invention to prepare transgenic plant。Such as, the polynucleotide of the present invention stably can be imported in the first department of botany that can carry out converting and (namely be integrated into genomic DNA) to generate transgenic plant, it is hybridized so that the polynucleotide of the present invention gradually penetrate in the second department of botany with the second department of botany。
Additionally provide and use the polynucleotide of the present invention to generate the method having enhancing character plant, said method comprising the steps of: the polynucleotide of the present invention are imported in host plant cell, select the cell that there is polynucleotide molecule to generate transgenic plant cells, and formed transgenic plant by transgenic plant cells regeneration, now compared to comparable wild-type plant or transgenic HaHB4 plant, transgenic plant has enhancing character, and wherein HaHB4 is under the control of same promoter。The method according to the invention the enhancing character of transgenic plant be can select, the water-use efficiency (such as " drought tolerance ") strengthened, the salt toleration strengthened, the osmotic pressure toleration of enhancing, the yield of raising and combination thereof included but not limited to。
Plant, seed and plant part product
In other embodiments, the present invention relates to production plant commodity and the method for producing plant commodity from plant described herein or plant part。Polynucleotide containing the present invention or the commodity of peptide sequence and the commodity produced by transgenic plant or the seed of the polynucleotide sequence containing the present invention are considered as embodiments of the present invention especially。Polynucleotide or the commodity of peptide sequence containing the present invention include but not limited to corn, oil, crushing or complete plant grain or seed or any recombinant plant grain comprising any corn, oil or crushing or the complete sequence containing one or more present invention or seed。Therefore, according to an embodiment, the present invention includes plant product after the polynucleotide of the present invention containing detectable amount or the processing of polypeptide, and wherein said plant product comprises the feedstuff of at least one part from plant, corn, flour, extract or homogenate。At another embodiment, the present invention includes plant product after the polynucleotide of the present invention containing detectable amount or the processing of polypeptide, and wherein said plant product comprises the feedstuff, corn, flour, extract or the homogenate that obtain from seed。The polynucleotide of the present invention contained in product after technology known in the art and reagent detection can be used to process and polypeptide, analyze including such as PCR and Northern, Southern and Western。
Gene stacking
Present invention additionally comprises the seed containing one or more transgenic events and plant。Present invention also contemplates that and can be used in combination the polynucleotide of the present invention and polypeptide and other transgenic " event " to produce with multiple required character or the plant strengthening character further。The transgenic event of these " superpositions " can relate to the event of same target organism or character, or relates to the event of different target pathogen, insecticide or character。Additionally, any means can be used to produce superposition event, include but not limited to that the cross-breeding of transgenic plant or multiple genetic convert。
In some embodiments, the transgenic seed of the present invention or plant band extraly are provided with the superposition transgenic event of herbicide tolerant。The example of the herbicide of recombinant expressed offer toleration includes but not limited to Mediben, grass fourth phosphine ammonium and glyphosate and N-((phosphonomethyl)) glycine (including its isopropyl amine salt form)。
In other embodiments, the transgenic seed of the present invention or plant band extraly are provided with the superposition transgenic event of insect-resistant。The recombinant expressed gene example providing insect-resistant includes but not limited to bacillus thuringiensis Cry variant (such as Cry1A, Cry1Ac, Cry2A, Cry1F-1Ac, Cry3A, Cry3Bb, Cry35Ab1) and/or Cyt gene family。
In other embodiments, the transgenic seed of the present invention or plant band extraly are provided with fungal disease, virus disease or bacterial disease or invade and harass the superposition transgenic event of (such as nematode infestation) resistance。
In other embodiments, the transgenic seed of the present invention or plant band extraly is provided with the superposition transgenic event of tolerance to environmental stress, and described environment-stress is selected from following group: drought condition, salt condition, osmotic pressure are coerced, low temperature exposes, beat exposure, nitrogen nutrient availability reduce, phosphorus nutrient availability reduces and high plant density。
In other embodiments, the transgenic seed of the present invention or plant are extraly with the superposition transgenic event making nitrogen service efficiency or output increased。
Embodiment
The present invention is explained further in the examples below。It should be understood that these embodiments following only provide by way of illustration。From what has been discussed above and following example, those skilled in the art can know the principal character of the present invention, it is possible to when without departing substantially from spirit and scope of the invention, the present invention carries out various change and improvement to be adapted to various application and condition。
Embodiment 1
Modified HaHB4.2 expresses the generation and qualification that build
The open reading frame that will be cloned into the cDNA in pBlueScriptSK-carrier (Si Cha column foot company (Stratagene), Uppsala, SWE) BamH1/Sac1 site encoding full leng HaHB4 (SEQIDNO:1) is used as the template of a series of PCR reaction to generate modified HaHB4。In the first step, use H4m-F forward primer (-ATGTCTCTTCAACAAGTAACAACCACCAGG-3'；And Transf2 reverse primer (5'-GCCGAGCTCTTAGAACTCCCACCACTTTTG-3' SEQIDNO:22)；SEQIDNO:23) PCR primer after PCR reaction generation first expands is carried out。Taking turns that the designed primer amplification of amplification obtains containing the transcriptional start site being newly introduced for this is the product of translational termination site。This first time pcr amplification product is cloned intoIn-T-Easy carrier (Pu Luomaige biotech company (PromegaBiotec), state of Wisconsin Madison) and called after " pTHaHB4.2a "。
Then pTHaHB4.2a crop template is used to use H4m-F forward primer (5'-ATGTCTCTTCAACAAGTAACAACCACCAGG-3'；And the reverse primer (5-TTAGAACTCCCACCACTTTTGAAGGTCTGG-3' of called after H4m-R SEQIDNO:22)；SEQIDNO:24) carry out second time pcr amplification reaction to generate second time pcr amplification product, be cloned into subsequentlyIn-T-Easy carrier and called after pTHaHB4.2b。
In third time pcr amplification reaction, using the template of pTHaHB4.2b crop PCR reaction and use two groups of primers, one group of primer is corresponding to Transf-1 forward primer (5'-GCGGGATCCACCATGTCTCTTCAACAAGTA-3'；And the reverse primer (5'-GTTTCCTTCTTCAAGGTACGCAAAACCGTCGC-3' of called after H4m-R1 SEQIDNO:26)；SEQIDNO:27), and second group of primer by H4m-F1 forward primer (5'-CGGTTTTGCGTACCTTGAAGAAGGAAACAGTTTG-3'；And reverse primer Transf-2 (5'-GCCGAGCTCTTAGAACTCCAACCACTTTTG-3' SEQIDNO:25)；SEQIDNO:23) composition。Use conventional recombinant techniques that the amplified production of these two groups of primers is fused to the chimeric polynucleotide sequence adjoined subsequently。Referring to such as SilverJ, LimjocoT, FeinstoneS (1995) Site-specificmutagenesisusingthepolymerasechainreaction (" using polymerase chain reaction to carry out mutation site-specific ")。In: InnisMA, GelfandDH, SninskyJJ edit, PCRStrategies (" PCR strategy "), company limited of academic press (AcademicPressInc), Santiago, 179-188 page。Briefly, this strategy includes two kinds of PCR primer are carried out degeneration, the step mixing subsequently and hybridizing。Use Klenow enzyme to extend hybrid product, use chimeric polynucleotide sequence crop template subsequently, use Transf-1 forward primer (SEQIDNO:26) and Transf-2 reverse primer (SEQIDNO:23) to carry out PCR reaction further。PCR primer after this reaction being expanded subsequently is cloned intoIn-T-Easy carrier and called after pTHaHB4.2c。
Express, at pTHaHB4.2c, each step building forming process and amplified production has been carried out sequence analysis, compared by the HaHB4 polynucleotide sequence with SEQIDNO:1, disclose following characteristics: after (a) amplification, PCR primer encodes a polypeptide, and it has four aminoacid deletion (the amino acid residue 7-10 of SEQIDNO:2) at amino terminal region；B the PCR primer after the amplification of () second time contains a P175L sudden change in the region in the HaHB4 activation structure territory of coding presumption, and have the disappearance of four nucleotide at 5'UTR place；C the product of () third time PCR reaction contains a L159F sudden change in the region of the carboxy terminal half of HaHB4；And the product after (d) above-mentioned last PCR reaction amplification comprises a conservative R22K sudden change。
The insertion in pTHaHB4.2c is expanded subsequently further to introduce conservative aminoacid change K22R in PCR reacts。Corresponding sequence of inserting in pTHaHB4.2c after amplification is checked order and determines peptide sequence (SEQIDNO:4) shown in its corresponding polynucleotide SEQIDNO:3 and code pattern 1A-B。The sequence alignment of Figure 1A-B shows the sequence difference between HaHB4 (SEQIDNO:2), HaHB4.2c (mod1HaHB4 (SEQIDNO:4)) and another modified HaHB4 transcription factor (mod2HaHB4 (SEQIDNO:8))。
HaHB4.2 expresses structure
By pTHaHB4.2c (HaHB4.2) polynucleotide sequence above generated by be cloned in the carrier of corn and soybean and Semen Tritici aestivi in the way of being operably connected with generate Fig. 2 A-2J the Semen sojae atricolor (Fig. 2 A-2C) that describes of systematicness, Semen Tritici aestivi (Fig. 2 D-2G) and Semen Maydis (Fig. 2 H-2J) express structure。Each structure of expressing described by Fig. 2 A-2J all comprises a HaHB4.2 sequence, it encodes 177 amino acid whose total length mod1HaHB4 (i.e. HaHB4.2 (SEQIDNO:4)) albumen, and with (a) constitutive promoter (such as 35SCaMV promoter (pZmHaHB4.2, pGmHaHB4.2) or Ubi promoter (pTaHaHB4.2)) or (b) inducible promoter (merged by the First Intron of modified HaHB4 promoter and arabidopsis Cox5c-2 gene (LPF-Cox) and formed) (pZmPrInHBH4.2, pGmPrInHB4.2, andpTaPrInHB4.2) it is operably connected。Each structure of expressing described by Fig. 2 A-2J also comprises the selected marker and a no terminator that are positioned at HaHB4.2cDNA downstream。
Embodiment 2
The production of modified HaHB4 transgenic plant
The generation of Semen sojae atricolor mod1HaHB4 transgenic event and selection
Agrobacterium-mediated transformation and Williams82 cultigen is used to produce mod1HaHB4 soybean transgenic event also。The three kinds of different expression cassettes reflecting a kind of composing type strategy and two kinds of induction type strategies are used to obtain the T1 of 35 independent events for seed。In composing type event, the expression of mod1HaHB4cDNA coded sequence is subject to 35SCaMV promoters driven。In induction type event, the expression of mod1HaHB4cDNA coded sequence is to be driven by the natural long allele of HaHB4 promoter or the chimeric polynucleotide sequence containing the identical long allele of HaHB4 promoter and AtCOX5c2 intron。
After conversion, the propagation first of cell carries out in greenhouse, and 10 T1 deriving from each event are easily separated test for individuality by PCR by period。Department of botany's (T1 separates for 3:1) sowing of the individual selfing of selected event will be derived from and plant is sampled, homozygous line is determined, it was shown that without negative segregant (each department of botany takes at least 5 individualities) in the offspring sampled by pcr analysis。The negative segregant of some identifying during screening is kept as comparison " blank system "。
Greenhouse carries out single copy homozygote and the seed expansion (T of blank system3For seed), period uses the seed of selected mod1HaHB4 homozygous line determine ethylene insensitivity and the expression of Hahb4 and downstream gene (LOX2 and CSD-1) in selected department of botany is carried out quantitatively。Also evaluate the selected mod1HaHB4 homozygous line tolerance for drought stress in laboratory conditions。Identify 22 strain list copy mod1HaHB4 homozygous cell lines for follow-up study。
The field test of modified HaHB-4 genetically engineered soybean
Argentina St. Louis province LiborioLuna area (33 ° of 35'15 " S, 65 ° 38 ' 09 " W), under field condition, soybean transgene (mod1HaHB4 (SEQIDNO:4)) and control series have been evaluated。Soil is sandy loam, pH5.99 and content of organics is 1.41%。Average annual rainfall is 800mm。
Planted 15 strain transgenic lines altogether with the ratio of every square metre of 28 strain plants, 7 strain feminine genders separate (blank) and are and wild type (Williams82)。Experimental design is completely random district group, subregion and carries out 3 repetitions, and for irrigation, sub-district is Semen sojae atricolor system in primary area。Employ two kinds of irrigation levels (low-level and high level)。For low-level irrigation method, suspend during plant propagation stage R1 to R6 and supply water。Therefore, which is only made up of twice water application, and once when the season of growth starts, another time is when the season of growth terminates。As shown in table 1, high level irrigation method is made up of the application of mensal water。
The long 5m of subregion, totally 4 row, interval 0.7m。Insecticide and fertilizer is used according to locality practice。Field data collection is arranged from the centre two of each subregion。Manual harvesting plant also uses ground fixing device to thrash。Have recorded seed weight and humidity。The yield of each repetition is listed in table 2。
Table 1: irrigation method during the field test season of growth and rainfall
Month Rainfall High level is irrigated Low-level is irrigated
January 80.52mm 115.0mm 115.0mm
February 65.02mm 112.0mm NA
March 51.05mm 130.0mm NA
April 6.1mm 20.0mm 20.0
Amount to 202.69mm 377.0mm 135.0mm
Using GLM method (SAS software) by subregion, field data to be analyzed, the method can difference between the data set of analytic band missing values。The standard of significant difference is alpha levels is 0.05。Process (high level and low-level irrigate) and be primary area and Semen sojae atricolor Xi Shizi district。Field data is in Table 2。
There is significant process (p=0.0079) and germline (p < 0.0001) main effect。Compared with high level irrigation method, low-level irrigation method causes that field yield declines 35% (from 2900Kgha-1Drop to 1897Kgha-1)。Owing to also not all homozygous line has corresponding blank, so having carried out the comparison between homozygous line and blank system building inside or between。The yield building the mod1HaHB4 homozygous line in inside or between and same system higher than blank is, a3H system is an exception。The yield of this homozygous line is blank system (a7N, a9N, b1N, b8N, b10N, c4N) lower than 6 kinds。For the inducible transgenic event in same system, between transgenic lines and blank system, it is absent from significant volume variance。But, the yield of composing type mod1HaHB4 transgenic event a7H and a9H is respectively higher than its blank, is all such under high level and low-level irrigation method。The yield that blank event a9N, a7N and isozygoty (a3H) are is substantially less than Williams82。
Table 2: low-level and high level irrigate the yield (kg.ha of the composing type mod1HaHB4 genetically engineered soybean system tested under growth conditions-1) data。(NA=without)。
Block diagram in Fig. 3 A-3D shows the raising of the genetically modified crops yield that (is not intended to supply water, be under non-water stress conditions) in high yield environment when the field through irrigating。Block diagram in Fig. 3 A-3B describes compared with the yield of wild type control Semen Maydis (WT), the raising of two kinds of homozygous transgenic Semen Maydis system's (expressing HaHB4.2 (SEQIDNO:4) under 35S constitutive promoter controls) yield。Data acquisition is from the parallel test point in two soil type areas: receives the sandy loam (Fig. 3 A) of 626mm rainfall between planting season and receives the well-drained sandy loam (Fig. 3 B) of 545mm rainfall in the crop cycle。Block diagram in Fig. 3 C describes compared with the yield of wild type control Semen Tritici aestivi (WT), the raising of a kind of homozygous transgenic Semen Tritici aestivi system's (expressing HaHB4.2 (SEQIDNO:4) under 35S constitutive promoter controls) yield。Data acquisition in Fig. 3 C is from the parallel test point of well-drained sandy loam area (pH7.14%, OM1.57%)。Use supplement irrigation to provide the water of 755mm within the crop cycle。Block diagram in Fig. 3 D describes compared with the yield of wild type control Semen sojae atricolor (WT), the raising of two kinds of homozygous transgenic Semen sojae atricolor system (expressing HaHB4.2 (SEQIDNO:4) under 35S constitutive promoter controls) yield。Data acquisition in Fig. 3 D is from the parallel test point of well-drained sandy loam area (pH5.99%, OM1.41%)。Use supplement irrigation to provide the water of 579mm within the crop cycle。
Identify from Semen Maydis system and Semen sojae atricolor system transgene method
Carry out Northern first by methods known in the art and material to analyze to determine the expression of HaHB4 in transgenic corns system and Semen sojae atricolor system。Use the regular-PCR method of general description herein and material that the corresponding transgene coding sequence of the HaHB4 that the isozygotys department of botany converted is expanded subsequently。Use methods known in the art to prepare Semen Maydis and soybean transgene genomic DNA, and be used as the template of PCR reaction。Use 35SPrimF forward primer (5'-TGACGCACAATCCCACTATC-3'(SEQIDNO:34)) and NOS121R reverse primer (5-GAATTCCCGATCTAGTAACATA-3'(SEQIDNO:35)) amplification composing type (35SCaMV promoter) the corresponding transgenic of expression cassette。Use TRANSF1Xba forward primer (5'-ATGTCTCTTCAACAAGTACCCAC-3'(SEQIDNO:32)), NOS121R reverse primer (5'-GAATTCCCGATCTAGTAACATA-3'(SEQIDNO:33)), and using Semen Maydis or soybean transgene genomic DNA as template, amplification induction type (TRANSF1Xba promoter) the corresponding transgenic of expression cassette in PCR reacts。
The sequence analysis that carries out of HaHB4 polynucleotide after amplification is identified the multiplexed differential between sequence and natural HaHB4 (SEQIDNO:1) sequence after amplification。Fig. 1 shows the sequence alignment coded by natural HaHB4 and mod1HaHB4 (HaHB4.2 (SEQIDNO:4)) and mod2HaHB4 (HaHB4.3 (SEQIDNO:8)) that this analysis identifies between sequence。
Embodiment 3
The trans-activation of modified HaHB4 in yeast
The polynucleotide encoding modified HaHB4 albumen (HaHB4.2 (mod1HaHB4), HaHB4.3 (mod3HaHB4) or HaHB4.4 (mod4HaHB4)) are cloned in pGBKT7 carrier and are operatively connected with the GAL4DNA binding structural domain in carrier, and measuring to convert in yeast one-hybrid is tested has gained to express the yeast built to determine the trans-activation of coded modified HaHB4。By using ONPG crop substrate to measure the betagalactosidase activity of the yeast containing modified HaHB4 albumen (with comparison HaHB4 albumen), it may be determined that whether modified HaHB4 (with comparison HaHB4 albumen) exists trans-activation。The block diagram of Fig. 4 shows that all modified HaHB4 albumen all can as activity factor in yeast one-hybrid pilot system。
Similarly, for determining that modified HaHB4 albumen forms the ability of heterodimer, Yeast two hybrid assay has been carried out。HaHB4.2cDNA it is cloned in pGAD carrier and merges with the activation structure territory of yeast transcription factor GAL4。Express gained subsequently to build and convert in saccharomyces cerevisiae AH109 strain, use express modified albumen and four kinds be cloned into pGBK7 carrier and the arabidopsis HD-Zip albumen (C end disappearance) with its upper GAL4 binding structural domain fusion carries out Yeast two hybrid assay。By the interaction that the auxotrophy evaluation in (-HIS) SD culture medium estimates。Result display HaHB4.2 can as transcription factor and can interact with AtHB1, AtHB7 and AtHB12。(data do not show)
These results show that the interaction between endogenous protein is probably mod1HaHB4 (HaHB4.2) and plays the Partial Mechanism of its function。
Embodiment 4
The form of transgenic Arabidopsis plants and photosynthesis rate
Impregnate (floraldip) technology by inflorescence to convert to arabidopsis thaliana 35S::H4.2 (including the expression structure of the mod1HaHB4 (SEQIDNO:4) being operably connected with 35S constitutive promoter) to produce transgenic Arabidopsis plants。Three strain plants planted by every basin, carry out blade CO2Gas exchange measures。The a piece of excised leaf of the plant every strain sampled is placed in the leaf sample pond of Licor6400XT photosynthesizer。By Licor6400LED light source irradiation blade, form about 1000 μm of olm-2s-1Photosynthetic photon flux density。
At leaf CO2Absorb steady Timing measurement photosynthesis rate (μm olm-2s-1), Licor6400 system fading margin enters the air in leaf sample pond to maintain temperature for about 24 DEG C and CO simultaneously2Concentration is about 500 μm of olmol-1。The block diagram of Fig. 5 shows the transgenic plant expressing empty carrier (pBI121) in two independent experiments and expresses the transgenic plant photosynthesis rate of mod1HaHB4 (HaHB4.2 the 30th is)。The photosynthesis rate that can be observed to express the transgenic plant of mod1HaHB4 is significantly higher than the check plant expressing empty carrier。This increases the output increased that may result in having converted the crop of modified mod1HaHB4。
This application claims the priority of the U.S. Provisional Application number 61/601,335 that on February 21st, 2012 submits to, it is incorporated herein by reference of text。Additionally, all publications cited herein are incorporated herein each through incorporated。
The description of above detailed description of the invention discloses the general aspects of the present invention completely, by applying the knowledge within the scope of art technology, other people without too much experiment can easily under the premise without departing substantially from general idea of the present invention just various using modified and/or revise these detailed description of the invention。Therefore, based on instruction listed herein and guide, it is thus understood that these amendments and improvement fall in implication and the scope of the equivalents of disclosed embodiment。Term or word in this specification it be to be understood that word herein and term are only illustrative and nonrestrictive, thus can be understood according to described instruction and guide by those skilled in the art。

Claims (54)

1. the nucleic acid molecules separated, the nucleic acid molecules of described separation is made up of the polynucleotide sequence encoding mod1HaHB4 shown in SEQIDNO:4。
2. the as claimed in claim 1 nucleic acid molecules separated, its coding is a kind of can in vitro in conjunction with the polypeptide of DNA。
3. the nucleic acid molecules separated as claimed in claim 1, it improves the yield of the plant having converted described nucleic acid molecules。
4. nucleic acid molecules as claimed in claim 1, it is operably connected with promoter。
5. nucleic acid molecules as claimed in claim 4, it is operably connected with constitutive promoter。
6. nucleic acid molecules as claimed in claim 5, described promoter is 35SCaMV promoter。
7. nucleic acid molecules as claimed in claim 5, described promoter is 35SCaMV promoter or Ub1 promoter。
8. nucleic acid molecules as claimed in claim 4, it is operably connected with inducible promoter。
9. nucleic acid molecules as claimed in claim 8, described promoter is HaHB4 promoter。
10. nucleic acid molecules as claimed in claim 9, the First Intron of described promoter and arabidopsis Cox5c2 merges。
11. a carrier, comprise the nucleic acid molecules described in claim 1。
12. carrier as claimed in claim 11, it also comprises the promoter being operably connected with described nucleic acid molecules。
13. carrier as claimed in claim 12, described vector expression is a kind of can in vitro in conjunction with the recombinant polypeptide of DNA。
14. a bacterial host cell, comprise the nucleic acid molecules described in claim 1。
15. a bacterial host cell, comprise the carrier described in claim 11。
16. a bacterial host cell, comprise the carrier described in claim 13。
17. a protein, it is characterised in that be the protein coded by the nucleic acid molecules described in claim 1。
18. a processed plant product, it comprises can polynucleotide described in the claim 1 of detected level。
19. plant product processed as claimed in claim 18, described product comprises and is derived from the feedstuff of at least one part of plant, meals, flour, extract or homogenate。
20. plant product processed as claimed in claim 18, described product comprises and is derived from the feedstuff of plant seed, meals, flour, extract or homogenate。
21. the method producing transgenic plant cells, described transgenic plant cells contains the nucleic acid molecules that the polynucleotide sequence of mod1HaHB4 shown in coding SEQIDNO:4 forms。
22. method as claimed in claim 21, the plant cell also including using Northern, Western, Southern or pcr analysis screening to convert is to determine that described plant cell comprises described nucleic acid molecules。
23. the method as described in claim 21, it is additionally included in described plant cell and expresses described polynucleotide sequence。
24. method as claimed in claim 21, described polynucleotide sequence is integrated in the DNA of described plant cell。
25. method as claimed in claim 21, described plant cell is monocot plant cell。
26. method as claimed in claim 25, described plant cell is maize cell。
27. method as claimed in claim 25, described plant cell is wheat cell。
28. method as claimed in claim 25, described plant cell is rice cell。
29. method as claimed in claim 21, described plant cell is dicotyledonous plant cells。
30. method as claimed in claim 29, described plant cell is soya cells。
31. method as claimed in claim 21, described polynucleotide sequence is operably connected with promoter。
32. method as claimed in claim 21, described plant cell expresses mod1HaHB4 shown in SEQIDNO:4。
33. method as claimed in claim 21, the offspring of described plant cell or described cell express a kind of can in vitro in conjunction with the recombiant protein of DNA。
34. the method as described in claim 21, it is additionally included in described transgenic plant cells and expresses described polynucleotide sequence。
35. the method as described in claim 21, also include described Plant cell regeneration is become plant。
36. the method producing high yield transformed host, described method comprises: use nucleic acid molecules stable conversion plant cell, described nucleic acid molecules is made up of the polynucleotide sequence encoding mod1HaHB4 shown in SEQIDNO:4, described nucleic acid can be expressed in described plant cell, and described cell regeneration is become plant。
37. method as claimed in claim 36, described plant cell is monocot plant cell。
38. method as claimed in claim 37, described plant cell is maize cell。
39. method as claimed in claim 37, described plant cell is wheat cell。
40. method as claimed in claim 37, described plant cell is rice cell。
41. method as claimed in claim 36, described plant cell is dicotyledonous plant cells。
42. method as claimed in claim 41, described plant cell is soya cells。
43. the method planting transgenic plant, described method comprises
A () plantation comprises nucleic acid according to any one of claim 1-10 or the transgenic seed of carrier according to any one of claim 11-13；With
B () grows into transgenic plant from described transgenic seed。
44. method as claimed in claim 43, described method also includes the step gathering in the crops described transgenic plant。
45. method as claimed in claim 44, described method also includes replanting the step of the seed of results from described transgenic plant。
46. the method as according to any one of claim 43-45, described plant is monocotyledon。
47. method as claimed in claim 46, described plant is Semen Maydis。
48. method as claimed in claim 46, described plant is Semen Tritici aestivi。
49. method as claimed in claim 46, described plant is Oryza sativa L.。
50. the method as according to any one of claim 43-45, described plant is dicotyledon。
51. method as claimed in claim 50, described plant is Semen sojae atricolor。
52. the nucleic acid according to any one of claim 1-10 or the carrier according to any one of claim 11-13 are for converting the purposes of plant cell。
53. the nucleic acid according to any one of claim 1-10, the carrier according to any one of claim 11-13 or the cell according to any one of claim 14-16 are for producing the purposes of transgenic plant。
54. purposes as claimed in claim 53, described transgenic plant contains the transgenic event of superposition。
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Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981729A (en) 1998-08-27 1999-11-09 Korea Kumho Petrochemical Co., Ltd. Transcription factor gene induced by water deficit and abscisic acid isolated from Arabidopsis thaliana
US6265638B1 (en) 1998-10-01 2001-07-24 Pioneer Hi-Bred International, Inc. Method of plant transformation
CA2767270A1 (en) 2000-04-07 2002-06-13 Basf Plant Science Gmbh Phosphatase stress-related proteins and methods of use in plants
WO2004099365A2 (en) 2003-05-02 2004-11-18 Lia Raquel Chan Transcription factor gene induced by water deficit conditions and abscisic acid from helianthus annuus, promoter and transgenic plants
US20080148432A1 (en) 2005-12-21 2008-06-19 Mark Scott Abad Transgenic plants with enhanced agronomic traits

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A cDNA Encoding an HD-Zip Protein from Sunflower;Raquel L，et al;《plant Physiol》;19941231;全文 *
G. M. GAGO,et al.Hahb-4，, a homeobox-leucine zipper gene potentially involved in abscisic acid-dependent responses to water stress in sunflower.《Plant, Cell and Environment》.2002,第25卷(第5期), *
Genbank.GenBank：AAA63768.2.《GenBank》.2001, *

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